The ability of brain cells to take in substances from their surface is essential to the production of a key ingredient in Alzheimer's brain plaques, neuroscientists at Washington University School of Medicine in St. Louis have learned.
The researchers used a drug to shut down the intake process, known as endocytosis, in a mouse model of Alzheimer's disease. The change led to a 70 percent drop in levels of amyloid beta, the protein fragment that clumps together to form Alzheimer's plaques. Importantly, they also found that endocytosis' ability to increase amyloid beta was coupled to normal nerve cell communication called synaptic activity
Blocking endocytosis isn't a viable option for treatment because cells throughout the body, including brain cells, need endocytosis for healthy function," says first author John Cirrito, Ph.D., research instructor in neurology. "But we are starting to understand the origins of amyloid beta in more detail now, and what we’re learning is opening other options we can pursue to seek new treatments for Alzheimer's disease
While endocytosis is necessary for normal function of brain cells, Cirrito and others believe it may accidentally be causing the cells to take in the amyloid precursor protein (APP), which breaks down into amyloid beta. If so, a drug that reduces brain cells' intake of APP may help reduce amyloid beta production.
The results appear in the April 10 issue of Neuron.
Other research had shown previously that endocytosis might be important for amyloid beta production, and that amyloid beta is produced inside brain cells. In 2005, Cirrito and his colleagues linked increased communication between brain cells to higher amyloid beta levels.
Cirrito decided to test both endocytosis and brain cell activity in a coordinated fashion. He used a technique known as microdialysis that he had previously adapted for Alzheimer's research to monitor the results. In addition to allowing repeated sampling of the amyloid beta levels in the brains of live mice, the approach allows him to introduce drugs that reduce endocytosis and alter communication between brain cells.
When researchers gave mice the drug that stopped endocytosis, amyloid beta levels dropped by 70 percent. To see how much normal brain activity contributed to ongoing amyloid beta production in the absence of endocytosis, they then added a second drug that reduced brain cell communication. Amyloid beta levels did not decrease further.
When they reversed the experiment, reducing brain cell communication first, amyloid beta decreased by 60 percent. Adding the drug that stops endocytosis caused an additional small reduction in amyloid beta.
The results show that amyloid beta production requires both brain cell communication and endocytosis, but endocytosis is essential for a slightly larger share of amyloid beta. Basic nerve cell physiology may explain why.
The study focused on synapses, the region where nerve cells transmit messages by releasing chemicals from small compartments near the cell surface. To replenish those compartments, the nerve cell regularly takes them back in through endocytosis. The more active a brain cell is, the more often it has to bring these compartments back into the cell and refill them.
Endocytosis can be messy in that it brings lots of substances into the cell from the membrane it internalizes," Cirrito says. "I think APP may be an innocent bystander in this process -- it just happens to be present on the cell surface when nerve cell communication causes more endocytosis. If there is a functional reason APP has to participate in this process, no one has found it."
Activity isn't the only cause of endocytosis in brain cells. The cells have other reasons for bringing in materials through endocytosis, and this additional intake could account for the small share of amyloid beta production that requires endocytosis but doesn't need brain cell activity.
Cirrito conducted the research in the laboratories of co-senior authors David M. Holtzman, M.D., the Andrew B. and Gretchen P. Jones Professor and chair of the Department of Neurology at the School of Medicine, and neurologist-in-chief at Barnes-Jewish Hospital, and Steven J. Mennerick, Ph.D., associate professor of neurobiology and psychiatry.
Researchers already know several proteins on the surfaces of brain cells that bind to APP. They will be conducting follow-up studies to see if blocking these interactions can block APP endocytosis and reduce amyloid beta production.
Saturday, April 12, 2008
Protecting a Life-Saving Blood Product from Human Form of Mad Cow Disease
Amid concern that recipients of certain blood transfusions may risk infection with a deadly protein responsible for the human form of mad cow disease, researchers in Canada now report development of a special filter that quickly and effectively removes the protein from blood.
In addition to causing mad cow disease, these so-called prion proteins cause a variant form of the human neurological disorder, Creutzfeldt-Jakob disease. Termed variant Creutzfeldt-Jakob Disease (vCJD), its emergence triggered recent bans on exportation of beef from Europe. Variant CJD also can be transmitted in blood transfusions
“The use of the device will significantly decrease the risk of acquiring vCJD through blood transfusions,” co-author Patrick V. Gurgel, Ph.D., reported at the 235th national meeting of the American Chemical Society. The device has been approved for use in Europe “and has no competitor at the moment,” said Gurgel.
About the size of a person’s hand, the device contains a specially-designed material that recognizes and binds to prions. “This technology adds a needed layer of protection against the transmission of vCJD through blood transfusion,” said Gurgel, senior research scientist at ProMetic Life Sciences in Mont-Royal, Quebec, Canada. “Our research shows that it works.”
The new filter can remove prions from red blood cell concentrate in less than an hour. Transfusions of red blood cells go to thousands of patients with chronic anemia resulting from kidney failure, cancer, gastrointestinal bleeding, and acute blood loss resulting from trauma.
The researchers needed five years to develop the device and are now working on ways to remove prion proteins from other blood components, including plasma and plasma proteins, Gurgel said.
In previous studies, the scientists showed that the device could successfully remove prions from the blood of infected hamsters and that the disinfected blood could be injected into healthy hamsters without causing disease. More recently, the researchers demonstrated that the device can also filter healthy human blood without damaging the red blood cells and other blood components, a finding that demonstrates that the technique is safe for use on human blood, they said.
Human clinical studies using the device, called the P-Capt® Prion Capture Filter, are now underway in Europe, where it has received approval for commercialization, the scientists say. The first commercialization will be in Ireland and the United Kingdom (UK) and is expected in mid 2008, in an effort to help safeguard blood supplies, Gurgel suggested
Experts believe that vCJD is acquired from eating beef from prion-infected cattle. As in cows, the disease is characterized by a slow destruction of the brain tissue, which results in nerve damage, paralysis, and eventually death. So far, vCJD has killed at least 200 people in Europe alone. Health officials are increasingly concerned that the disease may spread elsewhere, including the United States, through blood transfusions from infected individuals.
There is currently no reliable blood test for detecting the disease or a way of destroying the infectious prion proteins in blood. As a result, blood donation centers in the U.S. have imposed restrictions on blood donations from individuals who have lived in Europe for at least five years, particularly in the UK, where most vCJD cases have occurred.
In addition to causing mad cow disease, these so-called prion proteins cause a variant form of the human neurological disorder, Creutzfeldt-Jakob disease. Termed variant Creutzfeldt-Jakob Disease (vCJD), its emergence triggered recent bans on exportation of beef from Europe. Variant CJD also can be transmitted in blood transfusions
“The use of the device will significantly decrease the risk of acquiring vCJD through blood transfusions,” co-author Patrick V. Gurgel, Ph.D., reported at the 235th national meeting of the American Chemical Society. The device has been approved for use in Europe “and has no competitor at the moment,” said Gurgel.
About the size of a person’s hand, the device contains a specially-designed material that recognizes and binds to prions. “This technology adds a needed layer of protection against the transmission of vCJD through blood transfusion,” said Gurgel, senior research scientist at ProMetic Life Sciences in Mont-Royal, Quebec, Canada. “Our research shows that it works.”
The new filter can remove prions from red blood cell concentrate in less than an hour. Transfusions of red blood cells go to thousands of patients with chronic anemia resulting from kidney failure, cancer, gastrointestinal bleeding, and acute blood loss resulting from trauma.
The researchers needed five years to develop the device and are now working on ways to remove prion proteins from other blood components, including plasma and plasma proteins, Gurgel said.
In previous studies, the scientists showed that the device could successfully remove prions from the blood of infected hamsters and that the disinfected blood could be injected into healthy hamsters without causing disease. More recently, the researchers demonstrated that the device can also filter healthy human blood without damaging the red blood cells and other blood components, a finding that demonstrates that the technique is safe for use on human blood, they said.
Human clinical studies using the device, called the P-Capt® Prion Capture Filter, are now underway in Europe, where it has received approval for commercialization, the scientists say. The first commercialization will be in Ireland and the United Kingdom (UK) and is expected in mid 2008, in an effort to help safeguard blood supplies, Gurgel suggested
Experts believe that vCJD is acquired from eating beef from prion-infected cattle. As in cows, the disease is characterized by a slow destruction of the brain tissue, which results in nerve damage, paralysis, and eventually death. So far, vCJD has killed at least 200 people in Europe alone. Health officials are increasingly concerned that the disease may spread elsewhere, including the United States, through blood transfusions from infected individuals.
There is currently no reliable blood test for detecting the disease or a way of destroying the infectious prion proteins in blood. As a result, blood donation centers in the U.S. have imposed restrictions on blood donations from individuals who have lived in Europe for at least five years, particularly in the UK, where most vCJD cases have occurred.
Promising New Nanotechnology for Spinal Cord...
Promising New Nanotechnology for Spinal Cord Injury
A spinal cord injury often leads to permanent paralysis and loss of sensation below the site of the injury because the damaged nerve fibers can't regenerate. The nerve fibers or axons have the capacity to grow again, but don’t because they're blocked by scar tissue that develops around the injury.
Northwestern University researchers have shown that a new nano-engineered gel inhibits the formation of scar tissue at the injury site and enables the severed spinal cord fibers to regenerate and grow. The gel is injected as a liquid into the spinal cord and self -assembles into a scaffold that supports the new nerve fibers as they grow up and down the spinal cord, penetrating the site of the injury.
When the gel was injected into mice with a spinal cord injury, after six weeks the animals had a greatly enhanced ability to use their hind legs and walk.
The research is published today in the April 2 issue of the Journal of Neuroscience.
"We are very excited about this," said lead author John Kessler, M.D., Davee Professor of Stem Cell Biology at Northwestern University's Feinberg School of Medicine. "We can inject this without damaging the tissue. It has great potential for treating human beings.
"Kessler stressed caution, however, in interpreting the results. "It's important to understand that something that works in mice will not necessarily work in human beings. At this point in time we have no information about whether this would work in human beings."
"There is no magic bullet or one single thing that solves the spinal cord injury, but this gives us a brand new technology to be able to think about treating this disorder," said Kessler, also the chair of the Davee Department of Neurology at the Feinberg School. "It could be used in combination with other technologies including stem cells, drugs or other kinds of interventions."
“We designed our self-assembling nanostructures -- the building blocks of the gel -- to promote neuron growth,” said co-author Samuel I. Stupp, Board of Trustees Professor of Materials Science and Engineering, Chemistry, and Medicine and director of Northwestern’s Institute for BioNanotechnology in Medicine. “To actually see the regeneration of axons in the spinal cord after injury is a fascinating outcome.
”The nano-engineered gel works in several ways to support the regeneration of spinal cord nerve fibers. In addition to reducing the formation of scar tissue, it also instructs the stem cells --which would normally form scar tissue -- to instead to produce a helpful new cell that makes myelin. Myelin is a substance that sheaths the axons of the spinal cord to permit the rapid transmission of nerve impulses.
The gel's scaffolding also supports the growth of the axons in two critical directions -- up the spinal cord to the brain (the sensory axons) and down to the legs (the motor axons.) "Not everybody realizes you have to grow the fibers up the spinal cord so you can feel where the floor is. If you can't feel where the floor is with your feet, you can't walk," Kessler said.
Now Northwestern researchers are working on developing the nano-engineered gel to be acceptable as a pharmaceutical for the Food & Drug Administration.
If the gel is approved for humans, a clinical trial could begin in several years.
"It's a long way from helping a rodent to walk again and helping a human being walk again," Kessler stressed again. "People should never lose sight of that. But this is still exciting because it gives us a new technology for treating spinal cord injury."
A spinal cord injury often leads to permanent paralysis and loss of sensation below the site of the injury because the damaged nerve fibers can't regenerate. The nerve fibers or axons have the capacity to grow again, but don’t because they're blocked by scar tissue that develops around the injury.
Northwestern University researchers have shown that a new nano-engineered gel inhibits the formation of scar tissue at the injury site and enables the severed spinal cord fibers to regenerate and grow. The gel is injected as a liquid into the spinal cord and self -assembles into a scaffold that supports the new nerve fibers as they grow up and down the spinal cord, penetrating the site of the injury.
When the gel was injected into mice with a spinal cord injury, after six weeks the animals had a greatly enhanced ability to use their hind legs and walk.
The research is published today in the April 2 issue of the Journal of Neuroscience.
"We are very excited about this," said lead author John Kessler, M.D., Davee Professor of Stem Cell Biology at Northwestern University's Feinberg School of Medicine. "We can inject this without damaging the tissue. It has great potential for treating human beings.
"Kessler stressed caution, however, in interpreting the results. "It's important to understand that something that works in mice will not necessarily work in human beings. At this point in time we have no information about whether this would work in human beings."
"There is no magic bullet or one single thing that solves the spinal cord injury, but this gives us a brand new technology to be able to think about treating this disorder," said Kessler, also the chair of the Davee Department of Neurology at the Feinberg School. "It could be used in combination with other technologies including stem cells, drugs or other kinds of interventions."
“We designed our self-assembling nanostructures -- the building blocks of the gel -- to promote neuron growth,” said co-author Samuel I. Stupp, Board of Trustees Professor of Materials Science and Engineering, Chemistry, and Medicine and director of Northwestern’s Institute for BioNanotechnology in Medicine. “To actually see the regeneration of axons in the spinal cord after injury is a fascinating outcome.
”The nano-engineered gel works in several ways to support the regeneration of spinal cord nerve fibers. In addition to reducing the formation of scar tissue, it also instructs the stem cells --which would normally form scar tissue -- to instead to produce a helpful new cell that makes myelin. Myelin is a substance that sheaths the axons of the spinal cord to permit the rapid transmission of nerve impulses.
The gel's scaffolding also supports the growth of the axons in two critical directions -- up the spinal cord to the brain (the sensory axons) and down to the legs (the motor axons.) "Not everybody realizes you have to grow the fibers up the spinal cord so you can feel where the floor is. If you can't feel where the floor is with your feet, you can't walk," Kessler said.
Now Northwestern researchers are working on developing the nano-engineered gel to be acceptable as a pharmaceutical for the Food & Drug Administration.
If the gel is approved for humans, a clinical trial could begin in several years.
"It's a long way from helping a rodent to walk again and helping a human being walk again," Kessler stressed again. "People should never lose sight of that. But this is still exciting because it gives us a new technology for treating spinal cord injury."
Drug makers chase cancer stem cells
As evidence implicating stem cells in cancer mounts, drug makers are taking notice. GlaxoSmithKline (GSK) in December formed a strategic alliance worth up to $1.4 billion with OncoMed Pharmaceuticals, of Redwood City, California. The deal gives GSK an option to license four of OncoMed's antibody candidates developed to target cancer stem cells, one of which is scheduled to enter clinical trials in June.The GSK-OncoMed pact is the first major deal focused on cancer stem cell R&D, which is undergoing explosive growth. John Bates, the director of Biopharm Reports, in Cambridge, UK, says the number of companies devoted to this research has grown from 17 in April 2007 to nearly 40 today. What's more, patents covering developments in cancer stem cells doubled to about 70 in 2007, he adds. The problem is that not everyone even believes that targeting cancer stem cells will yield therapeutic benefits.
George Schreiner, CEO with Raven Biotechnologies in San Francisco, attributes the burst of commercial interest to recent evidence of cancer stem cells in solid tumors. Scientists have suspected since the 1950s that the cells play a role in blood tumors, such as acute myeloid leukemia, but their existence in solid tumors became evident only in 2003. That's when Michael Clarke, currently associate director of Stanford University's Institute for Stem Cell and Regenerative Medicine, and his then post-doc, Mohamed Al-Hajj, claimed to find cancer stem cells in breast tumors. The cells had two markers that are now synonymous with cancer stem cells: high expression of the antigen CD44 and low expression of antigen CD24. Isolated on the basis of these markers, the human cells were cultured and introduced into immunocompromised mice. Clarke and Al-Hajj found that only a few of the cells could spawn aggressive, metastatic tumors in the animals. Those findings bolstered a theory that solid tumors arise from a small population of cancer stem cells that, like normal stem cells, have the capacity for self-renewal. Clarke and his colleague Max Wicha, the director of the University of Michigan Comprehensive Cancer Center, founded OncoMed to pursue clinical opportunities in cancer stem cells in 2004. They now sit on the company's scientific advisory board.
Findings in other laboratories have since suggested cancer stem cells exist in various tumors, including those of the brain, head and neck, prostate, and colon. Scientists further postulate that cancer stem cells resist current drug therapies and repair DNA after radiation treatment more efficiently than their differentiated, daughter cells. That explains why solid tumors often recur after treatment, Schreiner explains. "What happens is the stem cells survive and repopulate to form a new tumor," he says. "And because they transmit their resistance to daughter cells, the new tumors are much harder to treat." Some researchers now believe the only way to cure cancer is by killing the stem cells that give rise to it.
OncoMed is one of a handful of companies preparing to test compounds against cancer stem cells in the clinic. In the GSK deal, OncoMed receives an undisclosed, up-front payment in cash and equity investment, with $1.4 billion more tied to achieving milestones. Royalties on product sales would follow. OncoMed's lead candidate, a humanized monoclonal antibody (mAb) OMP-21M18, targets "a cancer stem cell pathway with broad applicability across multiple solid tumors," says Paul Hastings, the company's CEO.
Other companies preparing for clinical trials this year include Arius Research in Toronto, whose lead humanized IgG1 mAb targets a variant form of CD44 found in leukemia, breast, colon and prostate cancer cells. Also, Raven Biotechnologies has two mAbs in preclinical development: RAV17 (which targets the pancreatic assigned tumor marker PAN), which Schreiner says targets prostate as well as pancreatic cancer cells, and RAV18 (which targets ADAM-9), for colon and lung cancer. Raven is now preparing to merge with VaxGen, a San Francisco-based vaccine manufacturer, picking up needed cash reserves from a company with a depleted pipeline but plenty of manufacturing assets. Reflecting a broader trend in cancer drug development, most compounds targeting cancer stem cells are monoclonal antibodies, Bates says (see Table 1). MAbs predominate because they target antigens on the cell surface rather than processes inside the cell as small molecules do.
The chief safety concern with targeting cancer stem cells, Clarke warns, is that these mAbs might also attack normal stem cells that replenish damaged tissues. "The main thing is to ensure that we eliminate the malignant cancer stem cells only without affecting the normal stem cells," he says. "Whether we'll be able to do this is the billion dollar question that everyone wants to answer."
Meanwhile, as commercial entities grow up around it, skeptics question the validity of targeting cancer stem cells. Current thinking holds that a tiny population of stem cells can explain why cancers recur even when existing treatments kill off up to 99% of a given tumor. According to Bert Volgestein, a professor of oncology at Johns Hopkins University in Baltimore, tumors can be completely eradicated only if those small—and presumably drug-resistant—stem cell fractions are destroyed.
The tumor fraction contributed by stem cells ranges from a low of 0.1% to a high of 40%, and some reports have described tumors made entirely of stem cells. But Vogelstein also admits that if a tumor containing a large fraction of stem cells were almost completely eliminated by treatment, this would undermine the logic of targeting stem cells as the last, drug-resistant holdouts from which aggressive metastatic tumors would likely emerge refractive to treatment.
GSK's interest in OncoMed comes from a desperation "to tap into oncology space, an area in which it is particularly weak," says Sho Matsubara, an analyst with London-based Standard and Poor's Equity Research Division. Also, GSK's sales are assumed to decline in coming years, due to generic competition (Matsubara estimates a 7% drop annually for the next five years). It does have a compound of its own that may have shrunk breast tumors by attacking cancer stem cells. According to evidence described at the San Antonio Breast Cancer Symposium on December 17, six weeks' treatment with GSK's Tyverb (lapatinib), a small molecule used in conjunction with Xeloda (capecitabine) for late-stage breast cancer, slashed the number of stem cells by more than half among 30 women studied. Two-thirds of the women were reportedly cancer-free after follow-up treatment.
But others remain cautious as, in some instances, claims pointing to the existence of cancer stem cells have turned out to be wrong upon closer inspection. "More studies are needed to confirm that cancer stem cells were in fact targeted by Tyverb," Bates notes. "We need further evidence to show that cancer stem cells in humans have been fully characterized. And we need ways to demonstrate that a particular subpopulation of cells has been reduced by treatment," he notes.
Ultimately, the best evidence will come from more studies that show killing cancer stem cells improves patient survival, Bates says. For fast-moving cancers such as pancreatic tumors, the evidence may come sooner. In the case of slow-moving cancers, such as prostate, accumulating the necessary evidence could take more time, he points out.
Monday, March 17, 2008
'To dream big has always been my motto': CEO at 27
Very few people can boast of achievements that M Thiagarajan, the 29-year-old promoter, chief executive officer and managing director of Chennai-based Paramount Airways notched so early in his life.
At 27, on October 19, 2005 to be precise, he launched India's first and only 'business class' airline Paramount Airways. Before that he had set up Paramount Mills in Madurai when he was dabbling in business management.
Apart from that he is an avid stargazer and a hobby pilot. In fact, an interesting incident inside a flight simulator in Frankfurt sparked Thiagarajan's passion for aviation.
"A pilot friend of mine had invited me to view his 747 Jumbo Jet flight simulator in Frankfurt. I was seated in the simulator and my friend was called away. I began to idly press the buttons and fidget with the controls. I created enough chaos to bring the flight instructor running!" said he in an e-mailed interview with rediff.com's Prasanna D Zore. That incident played a crucial role in Thiagarajan turning into an aviator.
Despite coming from an illustrious family -- his grandfather Sri Karumuttu Thiagaraja Chettiar, a reputed name in the field of textiles and education founded Bank of Madura which has now merged with ICICI Bank -- this gutsy aviator says that he believes in functioning independently of the family connections. "I believe that one needs to blaze one's own path in life."
And that he has done time and again to prove his point. When most entrepreneurs thought of starting low-cost airlines like Air Deccan and Spice Jet, Thiagarajan purchased the next-generation aircraft from Embraer to give travellers an 'Elite flying experience'.
The other feather in the cap of this 29-year-old is that Paramount succeeded in making operational profits within two years of its existence and boasts of an attrition rate of employees of zero per cent.
Very soon Paramount will spread its wingspan across India � currently it services only the southern sector � for the company is in the process of buying 40 more Embraer jets.
How did it all begin? Who and what inspired you to start an airline business? You had also started Paramount Mills before starting the airlines business.
What made you venture into the airline business?
I've always been fascinated by aviation and astronomy. Stargazing is a hobby and the wide expanse of the skies has always captivated me. My first encounter with aviation began when I was holidaying in Germany [Images], many years ago.
A pilot friend of mine had invited me to view his 747 Jumbo Jet flight simulator in Frankfurt. I was seated in the simulator and my friend was called away. I began to idly press the buttons and fidget with the controls. I created enough chaos to bring the flight instructor running!
But the incident, humourous though it was, sparked a very real passion for aviation. After that I started taking the pilot in me seriously and joined the flying school near London .When the instructor there asked me to fly a Cessna, I told him I only flew 747s. It was an amazing feeling to become a full-fledged pilot with a license to fly!
At 26, I wanted to nurture my passion further taking advantage of the Open Skies policy in India, I decided to set up Paramount Airways. To dream big has always been my motto and it was my desire to create an organisation that would reflect excellence and give me an opportunity to set up a global brand.
By then, I had already established my own textile mills -- Paramount Mills in Madurai, which won a national award for the highest export of cotton products from the Textile Export Promotion Council Of India. My journey while establishing Paramount Mills began even as I was graduating in management.
Having decided to venture into the civil aviation sector I started to assiduously research the various existing business models of international airline companies. This was to primarily understand the trend that was ingrained in commercial aviation. I soon realised that I didn't want to imitate any model.
I knew Paramount had to be unique; a model created to bring out the true joy of flying, to cherish and celebrate the experience of air travel. Paramount Airways is a 'High Value Carrier' -- one that gives you the best comfort in the skies at the best possible price, affording you true value for money and a flying experience that has no parallel.
You come from a very illustrious family. Did that help you when you launched Paramount Airways?
We're a very traditional family with three generations of involvement in the textile industry. My grandfather, Sri Karumuttu Thiagaraja Chettiar was a doyen in the field of textiles who founded several educational institutes and the former Bank of Madura which has now merged with ICICI Bank.
The family is rooted in ethical and traditional values, which include teetotalism and vegetarianism. I have a great passion for literature. As for my educational qualifications, I'm a business management graduate and a qualified hobby pilot.
My grandfather was an industrialist and philanthropist who had made great strides in business and education. He has always been my inspiration. However, I have always functioned independently of the family business; I believe that one needs to blaze one's own path in life.
Are there any advantages/disadvantages of starting an airline business at such an early age?
It is my firm belief that age is never a stumbling block as long as your vision is clear and your commitment to your goals is always your top priority.
Did you start this business on your own? What was the reaction of your family when you started this business?
Yes, Paramount Airways is my brainchild. My family has always been very supportive of the venture. Perhaps my family was a tad surprised that I was seriously considering another venture apart from textiles, over which the family has had a strong hold for generations.
What distinguishes your style of operations from the rest in the space? Who are your competitors?
Paramount Airways, translated literally means the pinnacle or the highest summit -- the peak of excellence.
Paramount Airways is a growing aspirational brand designed with the comfort of the traveller in mind. Our flight pattern is such that there is a convenient flight all through the day to all the sectors we fly. The day return flights have been well received by the business traveller as a great advantage. So we have been able to put together an innovative package of services and offer value based excellence to the discerning flyer.
This has fired the imagination of the customer who is value driven and appreciates a good brand when he sees one. We have differentiated ourselves as a value-based airline in terms of customer-orientation and have been categorised as a 'High Value Carrier' -- a model that is now being followed by other airlines across the world.
Can you share with us the secret behind Paramount Airways making operational profits with only two years into operations when other big airlines struggle to do it?
Dreaming big, a clear vision and sheer hard work makes the impossible, the often untried, happen!
How do you manage an attrition rate of zero per cent at Paramount Airways?
Quite simply -- we provide an excellent environment that nurtures and supports employees at all levels.
We have structured and well managed training programs, which, as a continuous improvement practice, is a great motivator and engages the employees while on the job. Apart from this, we involve each department in leisure pursuits outside of the office, in order for them to bond better and de-stress.
We also have established clear career paths bringing in technology and best international practices in our processes and systems. Our functional heads in key areas have international exposure, having run global airlines. All this, in addition to the right attitude in addressing employee related issues has minimised our attrition.
We also have specific employee recognition programs and send letters of commendation to those who excel at their tasks.
If somebody has to join Paramount Airways, what are the skills/qualities that you look out for in your potential staff?
I essentially look for the qualities of a winner -- someone who deeply believes in the company's ideals, would be willing to go that extra mile and are completely committed. We basically look for dynamic, creatively inspired individuals. Experience takes a back seat to attitude.
Any important message for young entrepreneurs who'd also want to make it big in life like you did?
My message to young entrepreneurs and readers is this -- persevere endlessly while believing in yourself and your dreams. Nurture your strength of mind and your power of conviction, while exploring our rich heritage and traditional roots. Live life to the fullest and contentment will always be yours!
What are your plans to make Paramount Airways the best airline in India/World?
Right now, the focus is on nurturing and further enhancing our loyal client base, providing as much of value as we can to the entire experience of flying to our 'Elite Travellers'.
I'm happy that Paramount has achieved global recognition for its unique model and has clearly established a leadership position in the Southern skies where we fly with a 26 per cent market share. What I am really proud of is the fact that Paramount through its unique offering and value-based service excellence has redefined airline travel in the country.
Concepts such as 'fine dining in the sky' with a four-course meal served in opulence and style on our aircraft and other fringe benefits such as valet service, 30 kgs baggage allowance (which is the highest when compared to the economy classes of other airlines), no cramped middle seats, the highest cabin crew per passenger ratio in the world on domestic sectors -- all translates into exceptional comfort and value.
Whether it is the gilded crockery or the linen serviette or the personal warm attention that our cabin crew gives to every passenger, our goal is to pamper our passengers. Every service we provide has been carefully tailor made for this. We take special care to ensure that our signature cuisine is created especially to suit our different clientele's tastes in various sectors that we fly.
A lot of care and effort has gone into seeing that a Paramount experience is a personal experience. We remain the first airline in the world to introduce an 'All Business Class' service bringing in quality differentiation in the basic offering itself. I have often heard people say that flying in a Paramount is akin to flying a corporate jet.
All this is a reflection of the business model that we arrived at after intensive research to understand the psyche of the typical passenger in the flying sector, his need for luxury, comfort and good service, the various business practices in the industry, even at the international level. Today we have largely succeeded in getting the discerning passenger to look at Paramount seriously primarily because of the fact that we are differentiated from the rest and appeal to his sense of belonging.
We have created a product that is aligned with the passengers' beliefs on moving up the ladder as per Maslow's theory of needs. With quality-edge and premium positioning we would like to establish our brand wherever we operate.
Winning the International Arch of Europe Award in the Gold Category in Frankfurt in February 2007 has helped reaffirm our goals. When I received the award from Mr Jose. E.Prieto, Executive President and CEO of the Business Initiatives Directions, he said and I quote "The awarded companies are symbols of commitment to leadership, technology and innovation which make them models for other companies in their sectors."
While the industry was focusing only on Airbus and Boeing we were the first to introduce the next-generation aircraft from Embraer which fly-by-wire. While giving the air-traveller a completely new experience we also made sure of our operational efficiency through this highly fuel efficient jets.
As for our future goals, we would like to saturate and consolidate our leadership position in the south and then get into the western India market which should happen shortly. We shall adopt the same strategy connecting Tier II and Tier III cities with the hub expanding and penetrating the market where possible.
We are in the process of acquiring 40 more Embraer jets over a period of time till 2011. Once we saturate and establish leadership in the western market we aim to have a National footprint by 2011.
At 27, on October 19, 2005 to be precise, he launched India's first and only 'business class' airline Paramount Airways. Before that he had set up Paramount Mills in Madurai when he was dabbling in business management.
Apart from that he is an avid stargazer and a hobby pilot. In fact, an interesting incident inside a flight simulator in Frankfurt sparked Thiagarajan's passion for aviation.
"A pilot friend of mine had invited me to view his 747 Jumbo Jet flight simulator in Frankfurt. I was seated in the simulator and my friend was called away. I began to idly press the buttons and fidget with the controls. I created enough chaos to bring the flight instructor running!" said he in an e-mailed interview with rediff.com's Prasanna D Zore. That incident played a crucial role in Thiagarajan turning into an aviator.
Despite coming from an illustrious family -- his grandfather Sri Karumuttu Thiagaraja Chettiar, a reputed name in the field of textiles and education founded Bank of Madura which has now merged with ICICI Bank -- this gutsy aviator says that he believes in functioning independently of the family connections. "I believe that one needs to blaze one's own path in life."
And that he has done time and again to prove his point. When most entrepreneurs thought of starting low-cost airlines like Air Deccan and Spice Jet, Thiagarajan purchased the next-generation aircraft from Embraer to give travellers an 'Elite flying experience'.
The other feather in the cap of this 29-year-old is that Paramount succeeded in making operational profits within two years of its existence and boasts of an attrition rate of employees of zero per cent.
Very soon Paramount will spread its wingspan across India � currently it services only the southern sector � for the company is in the process of buying 40 more Embraer jets.
How did it all begin? Who and what inspired you to start an airline business? You had also started Paramount Mills before starting the airlines business.
What made you venture into the airline business?
I've always been fascinated by aviation and astronomy. Stargazing is a hobby and the wide expanse of the skies has always captivated me. My first encounter with aviation began when I was holidaying in Germany [Images], many years ago.
A pilot friend of mine had invited me to view his 747 Jumbo Jet flight simulator in Frankfurt. I was seated in the simulator and my friend was called away. I began to idly press the buttons and fidget with the controls. I created enough chaos to bring the flight instructor running!
But the incident, humourous though it was, sparked a very real passion for aviation. After that I started taking the pilot in me seriously and joined the flying school near London .When the instructor there asked me to fly a Cessna, I told him I only flew 747s. It was an amazing feeling to become a full-fledged pilot with a license to fly!
At 26, I wanted to nurture my passion further taking advantage of the Open Skies policy in India, I decided to set up Paramount Airways. To dream big has always been my motto and it was my desire to create an organisation that would reflect excellence and give me an opportunity to set up a global brand.
By then, I had already established my own textile mills -- Paramount Mills in Madurai, which won a national award for the highest export of cotton products from the Textile Export Promotion Council Of India. My journey while establishing Paramount Mills began even as I was graduating in management.
Having decided to venture into the civil aviation sector I started to assiduously research the various existing business models of international airline companies. This was to primarily understand the trend that was ingrained in commercial aviation. I soon realised that I didn't want to imitate any model.
I knew Paramount had to be unique; a model created to bring out the true joy of flying, to cherish and celebrate the experience of air travel. Paramount Airways is a 'High Value Carrier' -- one that gives you the best comfort in the skies at the best possible price, affording you true value for money and a flying experience that has no parallel.
You come from a very illustrious family. Did that help you when you launched Paramount Airways?
We're a very traditional family with three generations of involvement in the textile industry. My grandfather, Sri Karumuttu Thiagaraja Chettiar was a doyen in the field of textiles who founded several educational institutes and the former Bank of Madura which has now merged with ICICI Bank.
The family is rooted in ethical and traditional values, which include teetotalism and vegetarianism. I have a great passion for literature. As for my educational qualifications, I'm a business management graduate and a qualified hobby pilot.
My grandfather was an industrialist and philanthropist who had made great strides in business and education. He has always been my inspiration. However, I have always functioned independently of the family business; I believe that one needs to blaze one's own path in life.
Are there any advantages/disadvantages of starting an airline business at such an early age?
It is my firm belief that age is never a stumbling block as long as your vision is clear and your commitment to your goals is always your top priority.
Did you start this business on your own? What was the reaction of your family when you started this business?
Yes, Paramount Airways is my brainchild. My family has always been very supportive of the venture. Perhaps my family was a tad surprised that I was seriously considering another venture apart from textiles, over which the family has had a strong hold for generations.
What distinguishes your style of operations from the rest in the space? Who are your competitors?
Paramount Airways, translated literally means the pinnacle or the highest summit -- the peak of excellence.
Paramount Airways is a growing aspirational brand designed with the comfort of the traveller in mind. Our flight pattern is such that there is a convenient flight all through the day to all the sectors we fly. The day return flights have been well received by the business traveller as a great advantage. So we have been able to put together an innovative package of services and offer value based excellence to the discerning flyer.
This has fired the imagination of the customer who is value driven and appreciates a good brand when he sees one. We have differentiated ourselves as a value-based airline in terms of customer-orientation and have been categorised as a 'High Value Carrier' -- a model that is now being followed by other airlines across the world.
Can you share with us the secret behind Paramount Airways making operational profits with only two years into operations when other big airlines struggle to do it?
Dreaming big, a clear vision and sheer hard work makes the impossible, the often untried, happen!
How do you manage an attrition rate of zero per cent at Paramount Airways?
Quite simply -- we provide an excellent environment that nurtures and supports employees at all levels.
We have structured and well managed training programs, which, as a continuous improvement practice, is a great motivator and engages the employees while on the job. Apart from this, we involve each department in leisure pursuits outside of the office, in order for them to bond better and de-stress.
We also have established clear career paths bringing in technology and best international practices in our processes and systems. Our functional heads in key areas have international exposure, having run global airlines. All this, in addition to the right attitude in addressing employee related issues has minimised our attrition.
We also have specific employee recognition programs and send letters of commendation to those who excel at their tasks.
If somebody has to join Paramount Airways, what are the skills/qualities that you look out for in your potential staff?
I essentially look for the qualities of a winner -- someone who deeply believes in the company's ideals, would be willing to go that extra mile and are completely committed. We basically look for dynamic, creatively inspired individuals. Experience takes a back seat to attitude.
Any important message for young entrepreneurs who'd also want to make it big in life like you did?
My message to young entrepreneurs and readers is this -- persevere endlessly while believing in yourself and your dreams. Nurture your strength of mind and your power of conviction, while exploring our rich heritage and traditional roots. Live life to the fullest and contentment will always be yours!
What are your plans to make Paramount Airways the best airline in India/World?
Right now, the focus is on nurturing and further enhancing our loyal client base, providing as much of value as we can to the entire experience of flying to our 'Elite Travellers'.
I'm happy that Paramount has achieved global recognition for its unique model and has clearly established a leadership position in the Southern skies where we fly with a 26 per cent market share. What I am really proud of is the fact that Paramount through its unique offering and value-based service excellence has redefined airline travel in the country.
Concepts such as 'fine dining in the sky' with a four-course meal served in opulence and style on our aircraft and other fringe benefits such as valet service, 30 kgs baggage allowance (which is the highest when compared to the economy classes of other airlines), no cramped middle seats, the highest cabin crew per passenger ratio in the world on domestic sectors -- all translates into exceptional comfort and value.
Whether it is the gilded crockery or the linen serviette or the personal warm attention that our cabin crew gives to every passenger, our goal is to pamper our passengers. Every service we provide has been carefully tailor made for this. We take special care to ensure that our signature cuisine is created especially to suit our different clientele's tastes in various sectors that we fly.
A lot of care and effort has gone into seeing that a Paramount experience is a personal experience. We remain the first airline in the world to introduce an 'All Business Class' service bringing in quality differentiation in the basic offering itself. I have often heard people say that flying in a Paramount is akin to flying a corporate jet.
All this is a reflection of the business model that we arrived at after intensive research to understand the psyche of the typical passenger in the flying sector, his need for luxury, comfort and good service, the various business practices in the industry, even at the international level. Today we have largely succeeded in getting the discerning passenger to look at Paramount seriously primarily because of the fact that we are differentiated from the rest and appeal to his sense of belonging.
We have created a product that is aligned with the passengers' beliefs on moving up the ladder as per Maslow's theory of needs. With quality-edge and premium positioning we would like to establish our brand wherever we operate.
Winning the International Arch of Europe Award in the Gold Category in Frankfurt in February 2007 has helped reaffirm our goals. When I received the award from Mr Jose. E.Prieto, Executive President and CEO of the Business Initiatives Directions, he said and I quote "The awarded companies are symbols of commitment to leadership, technology and innovation which make them models for other companies in their sectors."
While the industry was focusing only on Airbus and Boeing we were the first to introduce the next-generation aircraft from Embraer which fly-by-wire. While giving the air-traveller a completely new experience we also made sure of our operational efficiency through this highly fuel efficient jets.
As for our future goals, we would like to saturate and consolidate our leadership position in the south and then get into the western India market which should happen shortly. We shall adopt the same strategy connecting Tier II and Tier III cities with the hub expanding and penetrating the market where possible.
We are in the process of acquiring 40 more Embraer jets over a period of time till 2011. Once we saturate and establish leadership in the western market we aim to have a National footprint by 2011.
Friday, February 1, 2008
Could A Nanotube-based Drug Prevent Radiation Injury
The Department of Defense has commissioned a nine-month study from Rice University chemists and scientists in the Texas Medical Center to determine whether a new drug based on carbon nanotubes can help prevent people from dying of acute radiation injury following radiation exposure. The new study was commissioned after preliminary tests found the drug was greater than 5,000 times more effective at reducing the effects of acute radiation injury than the most effective drugs currently available.
"More than half of those who suffer acute radiation injury die within 30 days, not from the initial radioactive particles themselves but from the devastation they cause in the immune system, the gastrointestinal tract and other parts of the body," said James Tour, Rice's Chao Professor of Chemistry, director of Rice's Carbon Nanotechnology Laboratory (CNL) and principal investigator on the grant. "Ideally, we'd like to develop a drug that can be administered within 12 hours of exposure and prevent deaths from what are currently fatal exposure doses of ionizing radiation."
The Defense Advanced Research Projects Agency (DARPA) has awarded Tour and co-principal investigators J. Conyers and Valerie Moore at the University of Texas Health Science Center at Houston (UT-Houston) and Luka Milas, Kathy Mason and Jeffrey Myers at the University of Texas M.D. Anderson Cancer Center a $540,000 grant for a nine-month study of an experimental drug that the investigators have named Nanovector Trojan Horses (NTH).
NTH is made at Rice's Chemistry Department and Carbon Nanotechnology Laboratory in the Richard E. Smalley Institute for Nanoscale Science and Technology. The drug is based on single-walled carbon nanotubes, hollow cylinders of pure carbon that are about as wide as a strand of DNA. To form NTH, Rice scientists coat nanotubes with two common food preservatives -- the antioxidant compounds butylated hydroxyanisole (BHA) and butylated hydroxytoluene (BHT) -- and derivatives of those compounds.
"The same properties that make BHA and BHT good food preservatives, namely their ability to scavenge free radicals, also make them good candidates for mitigating the biological affects that are induced through the initial ionizing radiation event," Tour said.
In preliminary tests at M.D. Anderson in July 2007, mice showed enhanced protection when exposed to lethal doses of ionizing radiation when they were given first-generation NTH drugs prior to exposure.
"Our preliminary results are remarkable, and that's why DARPA awarded us this grant with a very compressed timeline for delivery: nine months, which is almost unheard of for an academic study of this type," Tour said. "They are very interested in finding out whether this will work in a post-exposure delivery, and they don't want to waste any time."
Ionizing radiation is any form of radioactive particle or energy that converts an atom or molecule into an ion by altering the balance between the number of protons and electrons. In living organisms, ionization often results in the creation of free radicals -- highly reactive molecules that can wreak havoc by disrupting healthy physiological processes. These free radicals induce a cascade of deleterious biological events that cause further destruction to the organism in the days and weeks after initial radiation exposure event. NTH is designed to terminate the destructive biological cascade.
Tour said the researchers are also interested in finding out whether the new drugs can prevent the unwanted side effects that cancer patients suffer after undergoing radiation therapy.
"More than half of those who suffer acute radiation injury die within 30 days, not from the initial radioactive particles themselves but from the devastation they cause in the immune system, the gastrointestinal tract and other parts of the body," said James Tour, Rice's Chao Professor of Chemistry, director of Rice's Carbon Nanotechnology Laboratory (CNL) and principal investigator on the grant. "Ideally, we'd like to develop a drug that can be administered within 12 hours of exposure and prevent deaths from what are currently fatal exposure doses of ionizing radiation."
The Defense Advanced Research Projects Agency (DARPA) has awarded Tour and co-principal investigators J. Conyers and Valerie Moore at the University of Texas Health Science Center at Houston (UT-Houston) and Luka Milas, Kathy Mason and Jeffrey Myers at the University of Texas M.D. Anderson Cancer Center a $540,000 grant for a nine-month study of an experimental drug that the investigators have named Nanovector Trojan Horses (NTH).
NTH is made at Rice's Chemistry Department and Carbon Nanotechnology Laboratory in the Richard E. Smalley Institute for Nanoscale Science and Technology. The drug is based on single-walled carbon nanotubes, hollow cylinders of pure carbon that are about as wide as a strand of DNA. To form NTH, Rice scientists coat nanotubes with two common food preservatives -- the antioxidant compounds butylated hydroxyanisole (BHA) and butylated hydroxytoluene (BHT) -- and derivatives of those compounds.
"The same properties that make BHA and BHT good food preservatives, namely their ability to scavenge free radicals, also make them good candidates for mitigating the biological affects that are induced through the initial ionizing radiation event," Tour said.
In preliminary tests at M.D. Anderson in July 2007, mice showed enhanced protection when exposed to lethal doses of ionizing radiation when they were given first-generation NTH drugs prior to exposure.
"Our preliminary results are remarkable, and that's why DARPA awarded us this grant with a very compressed timeline for delivery: nine months, which is almost unheard of for an academic study of this type," Tour said. "They are very interested in finding out whether this will work in a post-exposure delivery, and they don't want to waste any time."
Ionizing radiation is any form of radioactive particle or energy that converts an atom or molecule into an ion by altering the balance between the number of protons and electrons. In living organisms, ionization often results in the creation of free radicals -- highly reactive molecules that can wreak havoc by disrupting healthy physiological processes. These free radicals induce a cascade of deleterious biological events that cause further destruction to the organism in the days and weeks after initial radiation exposure event. NTH is designed to terminate the destructive biological cascade.
Tour said the researchers are also interested in finding out whether the new drugs can prevent the unwanted side effects that cancer patients suffer after undergoing radiation therapy.
Tuesday, January 29, 2008
RT-PCR: The Basics
RT-PCR (reverse transcription-polymerase chain reaction) is the most sensitive technique for mRNA detection and quantitation currently available. Compared to the two other commonly used techniques for quantifying mRNA levels, Northern blot analysis and RNase protection assay, RT-PCR can be used to quantify mRNA levels from much smaller samples. In fact, this technique is sensitive enough to enable quantitation of RNA from a single cell.
This article first discusses the advantages of real-time RT-PCR compared to end-point methods. This discussion is followed by a description of the different methods for quantitating gene expression by real-time RT-PCR with respect to the different chemistries available, the quantitation methods used and the instrumentation options available. Subsequently, the “traditional” methods of quantitating gene expression by RT-PCR, i.e. end-point techniques, are presented.
Why Real-Time RT-PCR?
Over the last several years, the development of novel chemistries and instrumentation platforms enabling detection of PCR products on a real-time basis has led to widespread adoption of real-time RT-PCR as the method of choice for quantitating changes in gene expression. Furthermore, real-time RT-PCR has become the preferred method for validating results obtained from array analyses and other techniques that evaluate gene expression changes on a global scale.To truly appreciate the benefits of real-time PCR, a review of PCR fundamentals is necessary.
At the start of a PCR reaction, reagents are in excess, template and product are at low enough concentrations that product renaturation does not compete with primer binding, and amplification proceeds at a constant, exponential rate. The point at which the reaction rate ceases to be exponential and enters a linear phase of amplification is extremely variable, even among replicate samples, but it appears to be primarily due to product renaturation competing with primer binding (since adding more reagents or enzyme has little effect). At some later cycle the amplification rate drops to near zero (plateaus), and little more product is made. For the sake of accuracy and precision, it is necessary to collect quantitative data at a point in which every sample is in the exponential phase of amplification (since it is only in this phase that amplification is extremely reproducible). Analysis of reactions during exponential phase at a given cycle number should theoretically provide several orders of magnitude of dynamic range. Rare targets will probably be below the limit of detection, while abundant targets will be past the exponential phase. In practice, a dynamic range of 2-3 logs can be quantitated during end-point relative RT-PCR. In order to extend this range, replicate reactions may be performed for a greater or lesser number of cycles, so that all of the samples can be analyzed in the exponential phase. Real-time PCR automates this otherwise laborious process by quantitating reaction products for each sample in every cycle.
The result is an amazingly broad 107-fold dynamic range, with no user intervention or replicates required. Data analysis, including standard curve generation and copy number calculation, is performed automatically. With increasing numbers of labs and core facilities acquiring the instrumentation required for real-time analysis, this technique is becoming the dominant RT-PCR-based quantitation technique.Real-Time PCR ChemistriesCurrently four different chemistries, TaqMan® (Applied Biosystems, Foster City, CA, USA), Molecular Beacons, Scorpions® and SYBR® Green (Molecular Probes), are available for real-time PCR. All of these chemistries allow detection of PCR products via the generation of a fluorescent signal. TaqMan probes, Molecular Beacons and Scorpions depend on Förster Resonance Energy Transfer (FRET) to generate the fluorescence signal via the coupling of a fluorogenic dye molecule and a quencher moeity to the same or different oligonucleotide substrates. SYBR Green is a fluorogenic dye that exhibits little fluorescence when in solution, but emits a strong fluorescent signal upon binding to double-stranded DNA.TaqMan ProbesTaqMan probes depend on the 5'- nuclease activity of the DNA polymerase used for PCR to hydrolyze an oligonucleotide that is hybridized to the target amplicon. TaqMan probes are oligonucleotides that have a fluorescent reporter dye attached to the 5' end and a quencher moeity coupled to the 3' end.
These probes are designed to hybridize to an internal region of a PCR product. In the unhybridized state, the proximity of the fluor and the quench molecules prevents the detection of fluorescent signal from the probe. During PCR, when the polymerase replicates a template on which a TaqMan probe is bound, the 5'- nuclease activity of the polymerase cleaves the probe. This decouples the fluorescent and quenching dyes and FRET no longer occurs. Thus, fluorescence increases in each cycle, proportional to the amount of probe cleavageWell-designed TaqMan probes require very little optimization. In addition, they can be used for multiplex assays by designing each probe with a spectrally unique fluor/quench pair. However, TaqMan probes can be expensive to synthesize, with a separate probe needed for each mRNA target being analyzed.Molecular BeaconsLike TaqMan probes, Molecular Beacons also use FRET to detect and quantitate the synthesized PCR product via a fluor coupled to the 5' end and a quench attached to the 3' end of an oligonucleotide substrate. Unlike TaqMan probes, Molecular Beacons are designed to remain intact during the amplification reaction, and must rebind to target in every cycle for signal measurement. Molecular Beacons form a stem-loop structure when free in solution.
Thus, the close proximity of the fluor and quench molecules prevents the probe from fluorescing. When a Molecular Beacon hybridizes to a target, the fluorescent dye and quencher are separated, FRET does not occur, and the fluorescent dye emits light upon irradiation. Molecular Beacons, like TaqMan probes, can be used for multiplex assays by using spectrally separated fluor/quench moieties on each probe. As with TaqMan probes, Molecular Beacons can be expensive to synthesize, with a separate probe required for each target.ScorpionsWith Scorpion probes, sequence-specific priming and PCR product detection is achieved using a single oligonucleotide. The Scorpion probe maintains a stem-loop configuration in the unhybridized state. The fluorophore is attached to the 5' end and is quenched by a moiety coupled to the 3' end. The 3' portion of the stem also contains sequence that is complementary to the extension product of the primer. This sequence is linked to the 5' end of a specific primer via a non-amplifiable monomer. After extension of the Scorpion primer, the specific probe sequence is able to bind to its complement within the extended amplicon thus opening up the hairpin loop. This prevents the fluorescence from being quenched and a signal is observed. SYBR GreenSYBR Green provides the simplest and most economical format for detecting and quantitating PCR products in real-time reactions. SYBR Green binds double-stranded DNA, and upon excitation emits light. Thus, as a PCR product accumulates, fluorescence increases.
The advantages of SYBR Green are that it is inexpensive, easy to use, and sensitive. The disadvantage is that SYBR Green will bind to any double-stranded DNA in the reaction, including primer-dimers and other non-specific reaction products, which results in an overestimation of the target concentration. For single PCR product reactions with well designed primers, SYBR Green can work extremely well, with spurious non-specific background only showing up in very late cycles.SYBR Green is the most economical choice for real-time PCR product detection. Since the dye binds to double-stranded DNA, there is no need to design a probe for any particular target being analyzed. However, detection by SYBR Green requires extensive optimization. Since the dye cannot distinguish between specific and non-specific product accumulated during PCR, follow up assays are needed to validate results.Real-time Reporters for Multiplex PCR TaqMan probes, Molecular Beacons and Scorpions allow multiple DNA species to be measured in the same sample (multiplex PCR), since fluorescent dyes with different emission spectra may be attached to the different probes. Multiplex PCR allows internal controls to be co-amplified and permits allele discrimination in single-tube, homogeneous assays.
These hybridization probes afford a level of discrimination impossible to obtain with SYBR Green, since they will only hybridize to true targets in a PCR and not to primer-dimers or other spurious products. Quantitation of ResultsTwo strategies are commonly employed to quantify the results obtained by real-time RT-PCR; the standard curve method and the comparative threshold method. These are discussed briefly below.Standard Curve MethodIn this method, a standard curve is first constructed from an RNA of known concentration. This curve is then used as a reference standard for extrapolating quantitative information for mRNA targets of unknown concentrations. Though RNA standards can be used, their stability can be a source of variability in the final analyses. In addition, using RNA standards would involve the construction of cDNA plasmids that have to be in vitro transcribed into the RNA standards and accurately quantitated, a time-consuming process. However, the use of absolutely quantitated RNA standards will help generate absolute copy number data. In addition to RNA, other nucleic acid samples can be used to construct the standard curve, including purified plasmid dsDNA, in vitro generated ssDNA or any cDNA sample expressing the target gene. Spectrophotometric measurements at 260 nm can be used to assess the concentration of these DNAs, which can then be converted to a copy number value based on the molecular weight of the sample used. cDNA plasmids are the preferred standards for standard curve quantitation. However, since cDNA plasmids will not control for variations in the efficiency of the reverse transcription step, this method will only yield information on relative changes in mRNA expression.
This, and variation introduced due to variable RNA inputs, can be corrected by normalization to a housekeeping gene.Comparative Ct Method Another quantitation approach is termed the comparative Ct method. This involves comparing the Ct values of the samples of interest with a control or calibrator such as a non-treated sample or RNA from normal tissue. The Ct values of both the calibrator and the samples of interest are normalized to an appropriate endogenous housekeeping gene. The comparative Ct method is also known as the 2–[delta][delta]Ct method, where [delta][delta]Ct = [delta]Ct,sample - [delta]Ct,referenceHere, [delta]CT,sample is the Ct value for any sample normalized to the endogenous housekeeping gene and [delta]Ct, reference is the Ct value for the calibrator also normalized to the endogenous housekeeping gene.For the [delta][delta]Ct calculation to be valid, the amplification efficiencies of the target and the endogenous reference must be approximately equal. This can be established by looking at how [delta]Ct varies with template dilution. If the plot of cDNA dilution versus delta Ct is close to zero, it implies that the efficiences of the target and housekeeping genes are very similar. If a housekeeping gene cannot be found whose amplification efficiency is similar to the target, then the standard curve method is preferred.Instrumentation for Real-Time PCR Real-time PCR requires an instrumentation platform that consists of a thermal cycler, a computer, optics for fluorescence excitation and emission collection, and data acquisition and analysis software. These machines, available from several manufacturers, differ in sample capacity (some are 96-well standard format, others process fewer samples or require specialized glass capillary tubes), method of excitation (some use lasers, others broad spectrum light sources with tunable filters), and overall sensitivity. There are also platform-specific differences in how the software processes data.
Real-time PCR machines are not inexpensive, currently about $25K - $95K, but are well within purchasing reach of core facilities or labs that have the need for high throughput quantitative analysis. For a comprehensive list of real-time thermal cyclers please see the weblink at the end of this article.Tools for Real-Time RT-PCRAmbion’s MessageSensor™ RT Kit includes an RNase H+ MMLV RT that clearly outperforms MMLV RT enzymes that have abolished RNase H activity in real-time RT-PCR experiments. Unlike many other qRT-PCR kits, MessageSensor includes a total RNA control, a control human GAPDH primer set, RNase inhibitor, and nucleotides, as well as a buffer additive that enables detection with SYBR® Green dye. The Cells-to-cDNA™ II Kit produces cDNA from cultured mammalian cells in less than 2 hours. No RNA isolation is required. This kit is ideal for those who want to perform reverse transcription reactions on small numbers of cells, numerous cell samples, or for scientists who are unfamiliar with RNA isolation. Ambion's Cells-to-cDNA II Kit contains a novel Cell Lysis Buffer that inactivates endogenous RNases without compromising downstream enzymatic reactions. After inactivation of RNases, the cell lysate can be directly added to a cDNA synthesis reaction. Cells-to-cDNA II is compatible with both one-step and two-step real-time RT-PCR protocols.Genomic DNA contamination can lead to false positive RT-PCR results. Ambion offers a variety of tools for eliminating genomic DNA contamination from RNA samples prior to RT-PCR.
Ambion’s DNA-free™ DNase Treatment and Removal Reagents are designed for removing contaminating DNA from RNA samples and for the removal of DNase after treatment without Proteinase K treatment and organic extraction. In addition, Ambion has also developed TURBO™ DNase, a hyperactive enzyme engineered from wild-type bovine DNase. The proficiency of TURBO DNase in binding very low concentrations of DNA means that the enzyme is particularly effective in removing trace quantities of DNA contamination.Ambion now also offers an economical alternative to the high cost of PCR reagents for the ABI 7700 and other 0.2 ml tube-based real-time instruments. SuperTaq™ Real-Time performs as well or better than the more expensive alternatives, and includes dNTPs and a Reaction Buffer optimized for SYBR Green, TaqMan, and Molecular Beacon chemistries.End-Point RT-PCR: Relative vs. Competitive vs. Comparative In spite of the rapid advances made in the area of real-time PCR detection chemistries and instrumentation, end-point RT-PCR still remains a very commonly used technique for measuring changes in gene-expression in small sample numbers.
End-point RT-PCR can be used to measure changes in expression levels using three different methods: relative, competitive and comparative. The most commonly used procedures for quantitating end-point RT-PCR results rely on detecting a fluorescent dye such as ethidium bromide, or quantitation of P32-labeled PCR product by a phosphorimager or, to a lesser extent, by scintillation counting.Relative quantitation compares transcript abundance across multiple samples, using a co-amplified internal control for sample normalization. Results are expressed as ratios of the gene-specific signal to the internal control signal.
This yields a corrected relative value for the gene-specific product in each sample. These values may be compared between samples for an estimate of the relative expression of target RNA in the samples; for example, 2.5-fold more IL-12 in sample 2 than in sample 1. Absolute quantitation, using competitive RT-PCR, measures the absolute amount (e.g., 5.3 x 105 copies) of a specific mRNA sequence in a sample. Dilutions of a synthetic RNA (identical in sequence, but slightly shorter than the endogenous target) are added to sample RNA replicates and are co-amplified with the endogenous target. The PCR product from the endogenous transcript is then compared to the concentration curve created by the synthetic "competitor RNA." Comparative RT-PCR mimics competitive RT-PCR in that target message from each RNA sample competes for amplification reagents within a single reaction, making the technique reliably quantitative. Because the cDNA from both samples have the same PCR primer binding site, one sample acts as a competitor for the other, making it unnecessary to synthesize a competitor RNA sequence.Both relative and competitive RT-PCR quantitation techniques require pilot experiments. In the case of relative RT-PCR, pilot experiments include selection of a quantitation method and determination of the exponential range of amplification for each mRNA under study.
For competitive RT-PCR, a synthetic RNA competitor transcript must be synthesized and used in pilot experiments to determine the appropriate range for the standard curve. Comparative RT-PCR yields similar sensitivity as relative and competitive RT-PCR, but requires significantly less optimization and does not require synthesis of a competitor.Relative RT-PCR Relative RT-PCR uses primers for an internal control that are multiplexed in the same RT-PCR reaction with the gene specific primers. Internal control and gene-specific primers must be compatible — that is, they must not produce additional bands or hybridize to each other. The expression of the internal control should be constant across all samples being analyzed. Then the signal from the internal control can be used to normalize sample data to account for tube-to-tube differences caused by variable RNA quality or RT efficiency, inaccurate quantitation or pipetting. Common internal controls include ß-actin and GAPDH mRNAs and 18S rRNA. Unlike Northerns and nuclease protection assays, where an internal control probe is simply added to the experiment, the use of internal controls in relative RT-PCR requires substantial optimization. For relative RT-PCR data to be meaningful, the PCR reaction must be terminated when the products from both the internal control and the gene of interest are detectable and are being amplified within exponential phase (see Determining Exponential Range in PCR).
Because internal control RNAs are typically constituitively expressed housekeeping genes of high abundance, their amplification surpasses exponential phase with very few PCR cycles. It is therefore difficult to identify compatible exponential phase conditions where the PCR product from a rare message is detectable. Detection methods with low sensitivity, like ethidium bromide staining of agarose gels, are therefore not recommended. Detecting a rare message while staying in exponential range with an abundant message can be achieved several ways: 1) by increasing the sensitivity of product detection, 2) by decreasing the amount of input template in the RT or PCR reactions and/or 3) by decreasing the number of PCR cycles. Ambion recommends using 18S rRNA as an internal control because it shows less variance in expression across treatment conditions than ß-actin and GAPDH. However, because of its abundance, it is difficult to detect the PCR product for rare messages in the exponential phase of amplification of 18S rRNA. Ambion's patented Competimer™ Technology solves this problem by attenuating the 18S rRNA signal even to the level of rare messages. Attenuation results from the use of competimers — primers identical in sequence to the functional 18S rRNA primers but that are "blocked" at their 3'-end and, thus, cannot be extended by PCR. Competimers and primers are mixed at various ratios to reduce the amount of PCR product generated from 18S rRNA. Figure 1 illustrates that 18S rRNA primers without competimers cannot be used as an internal control because the 18S rRNA amplification overwhelms that of clathrin (compare panels A and B). Mixing primers with competimers at a 3:7 ratio attenuates the 18S rRNA signal, making 18S rRNA a practical internal control (panel C).
Figure 1. Ambion's QuantumRNA™ Technology in Multiplex Quantitative RT-PCR using 18S rRNA as an Internal Control. RT-PCR reactions on brain, embryo, liver, and spleen total RNA using A) primers for clathrin, B) primers for clathrin and 18S, or C) primers for clathrin, 18S rRNA primers and 18S rRNA Competimers. Note that without Competimers, 18S cannot be used as an internal control because of its high abundance (B). Addition of Competimers (C) makes multiplex PCR possible, providing sample-to-sample relative quantitation.
Ambion's QuantumRNA 18S Internal Standards contain 18S rRNA primers and competimers designed to amplify 18S rRNA in all eukaryotes. The Universal 18S Internal Standards function across the broadest range of organisms including plants, animals and many protozoa. The Classic I and Classic II 18S Internal Standards can be used with any vertebrate RNA sample. All 18S Internal Standards work well in multiplex RT-PCR. These kits also include control RNA and an Instruction Manual detailing the series of experiments needed to make relative RT-PCR data significant. For those researchers who have validated ß-actin as an appropriate internal control for their system, the QuantumRNA ß-actin Internal Standards are available.
Competitive RT-PCR
Competitive RT-PCR precisely quantitates a message by comparing RT-PCR product signal intensity to a concentration curve generated by a synthetic competitor RNA sequence. The competitor RNA transcript is designed for amplification by the same primers and with the same efficiency as the endogenous target. The competitor produces a different-sized product so that it can be distinguished from the endogenous target product by gel analysis. The competitor is carefully quantitated and titrated into replicate RNA samples. Pilot experiments are used to find the range of competitor concentration where the experimental signal is most similar. Finally, the mass of product in the experimental samples is compared to the curve to determine the amount of a specific RNA present in the sample. Some protocols use DNA competitors or random sequences for competitive RT-PCR. These competitors do not effectively control for variations in the RT reaction or for the amplification efficiency of the specific experimental sequence, as do RNA competitors. See The Accuracy of Competitive RT-PCR Depends on Using the Right Exogenous Standard for a further discussion on competitor choice and design.
Comparative RT-PCR
While exquisitely sensitive, both relative and competitive methods of qRT-PCR have drawbacks. Relative RT-PCR requires extensive optimization to ensure that the PCR is terminated when both the gene of interest and an internal control are in the exponential phase of amplification. Competitive RT-PCR requires that an exogenous "competitor" be synthesized for each target to be analyzed. However, comparative RT-PCR achieves the same level of sensitivity as these standard methods of qRT-PCR, with significantly less optimization. Target mRNAs from 2 samples are assayed simultaneously, each serving as a competitor for the other, making it possible to compare the relative abundance of target between samples. Comparative RT-PCR is ideal for analyzing target genes discovered by screening methods such as array analysis and differential display.
Tools for Any RT-PCR Technique
Whether you choose to perform real-time, relative, competitive, or comparative RT-PCR, Ambion offers products to simplify your RT-PCR experiments and make the data more quantitative. In addition to the specific products described above, Ambion offers SuperTaq™ Polymerase, M-MLV Reverse Transcriptase, and RNase-free PCR tubes.
This article first discusses the advantages of real-time RT-PCR compared to end-point methods. This discussion is followed by a description of the different methods for quantitating gene expression by real-time RT-PCR with respect to the different chemistries available, the quantitation methods used and the instrumentation options available. Subsequently, the “traditional” methods of quantitating gene expression by RT-PCR, i.e. end-point techniques, are presented.
Why Real-Time RT-PCR?
Over the last several years, the development of novel chemistries and instrumentation platforms enabling detection of PCR products on a real-time basis has led to widespread adoption of real-time RT-PCR as the method of choice for quantitating changes in gene expression. Furthermore, real-time RT-PCR has become the preferred method for validating results obtained from array analyses and other techniques that evaluate gene expression changes on a global scale.To truly appreciate the benefits of real-time PCR, a review of PCR fundamentals is necessary.
At the start of a PCR reaction, reagents are in excess, template and product are at low enough concentrations that product renaturation does not compete with primer binding, and amplification proceeds at a constant, exponential rate. The point at which the reaction rate ceases to be exponential and enters a linear phase of amplification is extremely variable, even among replicate samples, but it appears to be primarily due to product renaturation competing with primer binding (since adding more reagents or enzyme has little effect). At some later cycle the amplification rate drops to near zero (plateaus), and little more product is made. For the sake of accuracy and precision, it is necessary to collect quantitative data at a point in which every sample is in the exponential phase of amplification (since it is only in this phase that amplification is extremely reproducible). Analysis of reactions during exponential phase at a given cycle number should theoretically provide several orders of magnitude of dynamic range. Rare targets will probably be below the limit of detection, while abundant targets will be past the exponential phase. In practice, a dynamic range of 2-3 logs can be quantitated during end-point relative RT-PCR. In order to extend this range, replicate reactions may be performed for a greater or lesser number of cycles, so that all of the samples can be analyzed in the exponential phase. Real-time PCR automates this otherwise laborious process by quantitating reaction products for each sample in every cycle.
The result is an amazingly broad 107-fold dynamic range, with no user intervention or replicates required. Data analysis, including standard curve generation and copy number calculation, is performed automatically. With increasing numbers of labs and core facilities acquiring the instrumentation required for real-time analysis, this technique is becoming the dominant RT-PCR-based quantitation technique.Real-Time PCR ChemistriesCurrently four different chemistries, TaqMan® (Applied Biosystems, Foster City, CA, USA), Molecular Beacons, Scorpions® and SYBR® Green (Molecular Probes), are available for real-time PCR. All of these chemistries allow detection of PCR products via the generation of a fluorescent signal. TaqMan probes, Molecular Beacons and Scorpions depend on Förster Resonance Energy Transfer (FRET) to generate the fluorescence signal via the coupling of a fluorogenic dye molecule and a quencher moeity to the same or different oligonucleotide substrates. SYBR Green is a fluorogenic dye that exhibits little fluorescence when in solution, but emits a strong fluorescent signal upon binding to double-stranded DNA.TaqMan ProbesTaqMan probes depend on the 5'- nuclease activity of the DNA polymerase used for PCR to hydrolyze an oligonucleotide that is hybridized to the target amplicon. TaqMan probes are oligonucleotides that have a fluorescent reporter dye attached to the 5' end and a quencher moeity coupled to the 3' end.
These probes are designed to hybridize to an internal region of a PCR product. In the unhybridized state, the proximity of the fluor and the quench molecules prevents the detection of fluorescent signal from the probe. During PCR, when the polymerase replicates a template on which a TaqMan probe is bound, the 5'- nuclease activity of the polymerase cleaves the probe. This decouples the fluorescent and quenching dyes and FRET no longer occurs. Thus, fluorescence increases in each cycle, proportional to the amount of probe cleavageWell-designed TaqMan probes require very little optimization. In addition, they can be used for multiplex assays by designing each probe with a spectrally unique fluor/quench pair. However, TaqMan probes can be expensive to synthesize, with a separate probe needed for each mRNA target being analyzed.Molecular BeaconsLike TaqMan probes, Molecular Beacons also use FRET to detect and quantitate the synthesized PCR product via a fluor coupled to the 5' end and a quench attached to the 3' end of an oligonucleotide substrate. Unlike TaqMan probes, Molecular Beacons are designed to remain intact during the amplification reaction, and must rebind to target in every cycle for signal measurement. Molecular Beacons form a stem-loop structure when free in solution.
Thus, the close proximity of the fluor and quench molecules prevents the probe from fluorescing. When a Molecular Beacon hybridizes to a target, the fluorescent dye and quencher are separated, FRET does not occur, and the fluorescent dye emits light upon irradiation. Molecular Beacons, like TaqMan probes, can be used for multiplex assays by using spectrally separated fluor/quench moieties on each probe. As with TaqMan probes, Molecular Beacons can be expensive to synthesize, with a separate probe required for each target.ScorpionsWith Scorpion probes, sequence-specific priming and PCR product detection is achieved using a single oligonucleotide. The Scorpion probe maintains a stem-loop configuration in the unhybridized state. The fluorophore is attached to the 5' end and is quenched by a moiety coupled to the 3' end. The 3' portion of the stem also contains sequence that is complementary to the extension product of the primer. This sequence is linked to the 5' end of a specific primer via a non-amplifiable monomer. After extension of the Scorpion primer, the specific probe sequence is able to bind to its complement within the extended amplicon thus opening up the hairpin loop. This prevents the fluorescence from being quenched and a signal is observed. SYBR GreenSYBR Green provides the simplest and most economical format for detecting and quantitating PCR products in real-time reactions. SYBR Green binds double-stranded DNA, and upon excitation emits light. Thus, as a PCR product accumulates, fluorescence increases.
The advantages of SYBR Green are that it is inexpensive, easy to use, and sensitive. The disadvantage is that SYBR Green will bind to any double-stranded DNA in the reaction, including primer-dimers and other non-specific reaction products, which results in an overestimation of the target concentration. For single PCR product reactions with well designed primers, SYBR Green can work extremely well, with spurious non-specific background only showing up in very late cycles.SYBR Green is the most economical choice for real-time PCR product detection. Since the dye binds to double-stranded DNA, there is no need to design a probe for any particular target being analyzed. However, detection by SYBR Green requires extensive optimization. Since the dye cannot distinguish between specific and non-specific product accumulated during PCR, follow up assays are needed to validate results.Real-time Reporters for Multiplex PCR TaqMan probes, Molecular Beacons and Scorpions allow multiple DNA species to be measured in the same sample (multiplex PCR), since fluorescent dyes with different emission spectra may be attached to the different probes. Multiplex PCR allows internal controls to be co-amplified and permits allele discrimination in single-tube, homogeneous assays.
These hybridization probes afford a level of discrimination impossible to obtain with SYBR Green, since they will only hybridize to true targets in a PCR and not to primer-dimers or other spurious products. Quantitation of ResultsTwo strategies are commonly employed to quantify the results obtained by real-time RT-PCR; the standard curve method and the comparative threshold method. These are discussed briefly below.Standard Curve MethodIn this method, a standard curve is first constructed from an RNA of known concentration. This curve is then used as a reference standard for extrapolating quantitative information for mRNA targets of unknown concentrations. Though RNA standards can be used, their stability can be a source of variability in the final analyses. In addition, using RNA standards would involve the construction of cDNA plasmids that have to be in vitro transcribed into the RNA standards and accurately quantitated, a time-consuming process. However, the use of absolutely quantitated RNA standards will help generate absolute copy number data. In addition to RNA, other nucleic acid samples can be used to construct the standard curve, including purified plasmid dsDNA, in vitro generated ssDNA or any cDNA sample expressing the target gene. Spectrophotometric measurements at 260 nm can be used to assess the concentration of these DNAs, which can then be converted to a copy number value based on the molecular weight of the sample used. cDNA plasmids are the preferred standards for standard curve quantitation. However, since cDNA plasmids will not control for variations in the efficiency of the reverse transcription step, this method will only yield information on relative changes in mRNA expression.
This, and variation introduced due to variable RNA inputs, can be corrected by normalization to a housekeeping gene.Comparative Ct Method Another quantitation approach is termed the comparative Ct method. This involves comparing the Ct values of the samples of interest with a control or calibrator such as a non-treated sample or RNA from normal tissue. The Ct values of both the calibrator and the samples of interest are normalized to an appropriate endogenous housekeeping gene. The comparative Ct method is also known as the 2–[delta][delta]Ct method, where [delta][delta]Ct = [delta]Ct,sample - [delta]Ct,referenceHere, [delta]CT,sample is the Ct value for any sample normalized to the endogenous housekeeping gene and [delta]Ct, reference is the Ct value for the calibrator also normalized to the endogenous housekeeping gene.For the [delta][delta]Ct calculation to be valid, the amplification efficiencies of the target and the endogenous reference must be approximately equal. This can be established by looking at how [delta]Ct varies with template dilution. If the plot of cDNA dilution versus delta Ct is close to zero, it implies that the efficiences of the target and housekeeping genes are very similar. If a housekeeping gene cannot be found whose amplification efficiency is similar to the target, then the standard curve method is preferred.Instrumentation for Real-Time PCR Real-time PCR requires an instrumentation platform that consists of a thermal cycler, a computer, optics for fluorescence excitation and emission collection, and data acquisition and analysis software. These machines, available from several manufacturers, differ in sample capacity (some are 96-well standard format, others process fewer samples or require specialized glass capillary tubes), method of excitation (some use lasers, others broad spectrum light sources with tunable filters), and overall sensitivity. There are also platform-specific differences in how the software processes data.
Real-time PCR machines are not inexpensive, currently about $25K - $95K, but are well within purchasing reach of core facilities or labs that have the need for high throughput quantitative analysis. For a comprehensive list of real-time thermal cyclers please see the weblink at the end of this article.Tools for Real-Time RT-PCRAmbion’s MessageSensor™ RT Kit includes an RNase H+ MMLV RT that clearly outperforms MMLV RT enzymes that have abolished RNase H activity in real-time RT-PCR experiments. Unlike many other qRT-PCR kits, MessageSensor includes a total RNA control, a control human GAPDH primer set, RNase inhibitor, and nucleotides, as well as a buffer additive that enables detection with SYBR® Green dye. The Cells-to-cDNA™ II Kit produces cDNA from cultured mammalian cells in less than 2 hours. No RNA isolation is required. This kit is ideal for those who want to perform reverse transcription reactions on small numbers of cells, numerous cell samples, or for scientists who are unfamiliar with RNA isolation. Ambion's Cells-to-cDNA II Kit contains a novel Cell Lysis Buffer that inactivates endogenous RNases without compromising downstream enzymatic reactions. After inactivation of RNases, the cell lysate can be directly added to a cDNA synthesis reaction. Cells-to-cDNA II is compatible with both one-step and two-step real-time RT-PCR protocols.Genomic DNA contamination can lead to false positive RT-PCR results. Ambion offers a variety of tools for eliminating genomic DNA contamination from RNA samples prior to RT-PCR.
Ambion’s DNA-free™ DNase Treatment and Removal Reagents are designed for removing contaminating DNA from RNA samples and for the removal of DNase after treatment without Proteinase K treatment and organic extraction. In addition, Ambion has also developed TURBO™ DNase, a hyperactive enzyme engineered from wild-type bovine DNase. The proficiency of TURBO DNase in binding very low concentrations of DNA means that the enzyme is particularly effective in removing trace quantities of DNA contamination.Ambion now also offers an economical alternative to the high cost of PCR reagents for the ABI 7700 and other 0.2 ml tube-based real-time instruments. SuperTaq™ Real-Time performs as well or better than the more expensive alternatives, and includes dNTPs and a Reaction Buffer optimized for SYBR Green, TaqMan, and Molecular Beacon chemistries.End-Point RT-PCR: Relative vs. Competitive vs. Comparative In spite of the rapid advances made in the area of real-time PCR detection chemistries and instrumentation, end-point RT-PCR still remains a very commonly used technique for measuring changes in gene-expression in small sample numbers.
End-point RT-PCR can be used to measure changes in expression levels using three different methods: relative, competitive and comparative. The most commonly used procedures for quantitating end-point RT-PCR results rely on detecting a fluorescent dye such as ethidium bromide, or quantitation of P32-labeled PCR product by a phosphorimager or, to a lesser extent, by scintillation counting.Relative quantitation compares transcript abundance across multiple samples, using a co-amplified internal control for sample normalization. Results are expressed as ratios of the gene-specific signal to the internal control signal.
This yields a corrected relative value for the gene-specific product in each sample. These values may be compared between samples for an estimate of the relative expression of target RNA in the samples; for example, 2.5-fold more IL-12 in sample 2 than in sample 1. Absolute quantitation, using competitive RT-PCR, measures the absolute amount (e.g., 5.3 x 105 copies) of a specific mRNA sequence in a sample. Dilutions of a synthetic RNA (identical in sequence, but slightly shorter than the endogenous target) are added to sample RNA replicates and are co-amplified with the endogenous target. The PCR product from the endogenous transcript is then compared to the concentration curve created by the synthetic "competitor RNA." Comparative RT-PCR mimics competitive RT-PCR in that target message from each RNA sample competes for amplification reagents within a single reaction, making the technique reliably quantitative. Because the cDNA from both samples have the same PCR primer binding site, one sample acts as a competitor for the other, making it unnecessary to synthesize a competitor RNA sequence.Both relative and competitive RT-PCR quantitation techniques require pilot experiments. In the case of relative RT-PCR, pilot experiments include selection of a quantitation method and determination of the exponential range of amplification for each mRNA under study.
For competitive RT-PCR, a synthetic RNA competitor transcript must be synthesized and used in pilot experiments to determine the appropriate range for the standard curve. Comparative RT-PCR yields similar sensitivity as relative and competitive RT-PCR, but requires significantly less optimization and does not require synthesis of a competitor.Relative RT-PCR Relative RT-PCR uses primers for an internal control that are multiplexed in the same RT-PCR reaction with the gene specific primers. Internal control and gene-specific primers must be compatible — that is, they must not produce additional bands or hybridize to each other. The expression of the internal control should be constant across all samples being analyzed. Then the signal from the internal control can be used to normalize sample data to account for tube-to-tube differences caused by variable RNA quality or RT efficiency, inaccurate quantitation or pipetting. Common internal controls include ß-actin and GAPDH mRNAs and 18S rRNA. Unlike Northerns and nuclease protection assays, where an internal control probe is simply added to the experiment, the use of internal controls in relative RT-PCR requires substantial optimization. For relative RT-PCR data to be meaningful, the PCR reaction must be terminated when the products from both the internal control and the gene of interest are detectable and are being amplified within exponential phase (see Determining Exponential Range in PCR).
Because internal control RNAs are typically constituitively expressed housekeeping genes of high abundance, their amplification surpasses exponential phase with very few PCR cycles. It is therefore difficult to identify compatible exponential phase conditions where the PCR product from a rare message is detectable. Detection methods with low sensitivity, like ethidium bromide staining of agarose gels, are therefore not recommended. Detecting a rare message while staying in exponential range with an abundant message can be achieved several ways: 1) by increasing the sensitivity of product detection, 2) by decreasing the amount of input template in the RT or PCR reactions and/or 3) by decreasing the number of PCR cycles. Ambion recommends using 18S rRNA as an internal control because it shows less variance in expression across treatment conditions than ß-actin and GAPDH. However, because of its abundance, it is difficult to detect the PCR product for rare messages in the exponential phase of amplification of 18S rRNA. Ambion's patented Competimer™ Technology solves this problem by attenuating the 18S rRNA signal even to the level of rare messages. Attenuation results from the use of competimers — primers identical in sequence to the functional 18S rRNA primers but that are "blocked" at their 3'-end and, thus, cannot be extended by PCR. Competimers and primers are mixed at various ratios to reduce the amount of PCR product generated from 18S rRNA. Figure 1 illustrates that 18S rRNA primers without competimers cannot be used as an internal control because the 18S rRNA amplification overwhelms that of clathrin (compare panels A and B). Mixing primers with competimers at a 3:7 ratio attenuates the 18S rRNA signal, making 18S rRNA a practical internal control (panel C).
Figure 1. Ambion's QuantumRNA™ Technology in Multiplex Quantitative RT-PCR using 18S rRNA as an Internal Control. RT-PCR reactions on brain, embryo, liver, and spleen total RNA using A) primers for clathrin, B) primers for clathrin and 18S, or C) primers for clathrin, 18S rRNA primers and 18S rRNA Competimers. Note that without Competimers, 18S cannot be used as an internal control because of its high abundance (B). Addition of Competimers (C) makes multiplex PCR possible, providing sample-to-sample relative quantitation.
Ambion's QuantumRNA 18S Internal Standards contain 18S rRNA primers and competimers designed to amplify 18S rRNA in all eukaryotes. The Universal 18S Internal Standards function across the broadest range of organisms including plants, animals and many protozoa. The Classic I and Classic II 18S Internal Standards can be used with any vertebrate RNA sample. All 18S Internal Standards work well in multiplex RT-PCR. These kits also include control RNA and an Instruction Manual detailing the series of experiments needed to make relative RT-PCR data significant. For those researchers who have validated ß-actin as an appropriate internal control for their system, the QuantumRNA ß-actin Internal Standards are available.
Competitive RT-PCR
Competitive RT-PCR precisely quantitates a message by comparing RT-PCR product signal intensity to a concentration curve generated by a synthetic competitor RNA sequence. The competitor RNA transcript is designed for amplification by the same primers and with the same efficiency as the endogenous target. The competitor produces a different-sized product so that it can be distinguished from the endogenous target product by gel analysis. The competitor is carefully quantitated and titrated into replicate RNA samples. Pilot experiments are used to find the range of competitor concentration where the experimental signal is most similar. Finally, the mass of product in the experimental samples is compared to the curve to determine the amount of a specific RNA present in the sample. Some protocols use DNA competitors or random sequences for competitive RT-PCR. These competitors do not effectively control for variations in the RT reaction or for the amplification efficiency of the specific experimental sequence, as do RNA competitors. See The Accuracy of Competitive RT-PCR Depends on Using the Right Exogenous Standard for a further discussion on competitor choice and design.
Comparative RT-PCR
While exquisitely sensitive, both relative and competitive methods of qRT-PCR have drawbacks. Relative RT-PCR requires extensive optimization to ensure that the PCR is terminated when both the gene of interest and an internal control are in the exponential phase of amplification. Competitive RT-PCR requires that an exogenous "competitor" be synthesized for each target to be analyzed. However, comparative RT-PCR achieves the same level of sensitivity as these standard methods of qRT-PCR, with significantly less optimization. Target mRNAs from 2 samples are assayed simultaneously, each serving as a competitor for the other, making it possible to compare the relative abundance of target between samples. Comparative RT-PCR is ideal for analyzing target genes discovered by screening methods such as array analysis and differential display.
Tools for Any RT-PCR Technique
Whether you choose to perform real-time, relative, competitive, or comparative RT-PCR, Ambion offers products to simplify your RT-PCR experiments and make the data more quantitative. In addition to the specific products described above, Ambion offers SuperTaq™ Polymerase, M-MLV Reverse Transcriptase, and RNase-free PCR tubes.
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