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This RNA-amplification protocol uses "terminal continuation" primers to enable production of either the standard antisense strand or the novel sense strand (shown). (From: S. Che, S.D. Ginsberg,
Audrey Player is searching for the ideal RNA amplification protocol. In her microarray facility at the National Cancer Institute's Advanced Technology Center, she plans to genetically profile various subtypes of tumor cells obtained by laser-capture microdissection. The goal: to identify new targets for anticancer therapies.
But before she can begin, Player must find a protocol that can reliably amplify as little as 10 picograms of total input RNA from a single cell to the 5 or 20 micrograms necessary for microarray analysis (an increase of 107- to 108-fold) without distorting the original abundance ratios of each mRNA species. "Perhaps the most challenging aspect of studying the biology of small sample preparations or individual cells – what I like to call the ultimate homogenous sample – involves amplification of the RNA for application on the microarray," Player says.
About a dozen companies sell off-the-shelf RNA amplification kits, and Player has tested nearly all of them. Many of these can reliably amplify as little as one nanogram of input RNA, but when challenged to amplify 10 to 100 picograms (the amount obtained from one to 10 cells), Player says, "They are more likely to make junk. It's an extraordinarily ambitious goal."
Microarray analysis has become a valuable tool not only for cancer researchers, but also for neuroscientists, developmental biologists, and immunologists. For the busy researcher, obtaining reliable information about amplification kits – i.e., more than the glowing reviews provided in the product brochure – can be a difficult task.
MAJOR AMPLIFICATION TECHNIQUES
RNA amplification kits and protocols fall into two broad categories: those based on linear amplification and those based on PCR.
Linear amplification produces high-fidelity results that remain true to the original RNA ratios, but the method is not robust. The linear method can produce, at best, about 103-fold amplification, not enough for use with most microarray platforms. Additional rounds of amplification are often necessary, which take additional time and can degrade the sample.
PCR, on the other hand, can generate about a million-fold amplification in 20 cycles. But the PCR method tends to amplify different lengths of cDNA unequally, causing distorted results. (Player has ruled out PCR-based methods for her project.)
Of the many RNA-amplification techniques available, the linear Eberwine method is one of the most-widely used. The method underlies a number of commercial kits, and Sangdun Choi in the division of biology at the California Institute of Technology has tested several of them, including the cDNA Synthesis kits by Affymetrix of Santa Clara, Calif., the T7 kit by Ambion of Austin, Texas, and the linear amplification kit by Agilent Technologies of Palo Alto, Calif. Choi's lab also develops its own protocols. "The Eberwine method-based kits are producing relatively stable and reproducible results," he says.
In the Eberwine method, the first strand of cDNA is synthesized from mRNA using an oligo(dT) primer modified with a T7 promoter sequence. After the second strand is generated, the double-stranded cDNA becomes the template for antisense RNA synthesis using T7 RNA polymerase.
Two linear methods also employ T7 polymerase but are significantly different from the Eberwine method: the SenseAmp method by Genisphere of Hatfield, Pa., and the Ribo-SPIA method by NuGEN Technologies of San Carlos, Calif. Also of note, the BD SMART method from BD Biosciences-Clontech of Palo Alto combines T7 RNA polymerase transcription with PCR amplification. More complete technical details of these methods and others are available in the literature.1
DOING IT YOURSELF
When talking with scientists who use RNA amplification kits, it is difficult to find anyone who is enthusiastic about them. Many researchers say the ideal kit has yet to be created. Player, for instance, cites several shortcomings, including sample loss, an emphasis on speed and simplicity rather than yield, and poor sensitivity. "Most [kits] require at least one nanogram total input RNA," she says. "Those claiming higher sensitivity tend not to be reproducible."
Player is one of a small number of scientists who have tried to improve on existing kits by developing their own protocols; Stephen D. Ginsberg is another. A researcher at the Center for Dementia Research, New York University School of Medicine in Orangeburg, Ginsberg profiles cells from mouse and human postmortem brain tissue. He has had difficulties with commercial kits and currently does not use any. "We have developed our own RNA amplification method and are very happy with it," he says.
Although developing and following one's own protocol is cheaper than purchasing a kit, Ginsberg says, "The downside is that this process is not nearly as convenient; one has to synthesize primers, order enzymes from different sources, and keep track of all this stuff." He says he may develop his technology, called terminal continuation RNA amplification, into a kit. " [We] are currently looking for potential licensees."
Fredrik Kamme, of Johnson & Johnson Pharmaceutical Research and Development in San Diego, has also developed a functional RNA amplification system, which he and his colleagues licensed to Epicentre. The Madison, Wis.-based biotech firm commercialized Kamme's method in its TargetAmp series of amplification kits. "As far as I know, there are no published protocols more efficient than some of the commercial kits," Kamme says. "Certainly you can make it cheaper, but you have to invest the time to optimize the protocol and QC reagents. It's important to realize that the T7 amplification protocols have to be finely tuned for optimal performance."
Most scientists do not have the time or the resources – or, for that matter, the inclination – to create their own protocols and must therefore rely on commercial kits. "In the past, we did set up our custom-made amplification reactions, including the implementation of some modifications of the Baugh method," says Alfonso Bellacosa, of the Fox Chase Cancer Center in Philadelphia, who is analyzing colorectal epithelial cells. The Baugh method is another linear, T7-based protocol. "However, at this point," he says, "we only use commercial kits," including the Ovation nanosample RNA amplification system from NuGEN of San Carlos, Calif., and the RiboAmp RNA amplification kit from Arcturus of Mountain View, Calif. "They are easier to work with and hopefully more controlled and standardized. This is what offsets the cost at this point."
The cost can indeed be substantial. Amplification kits range from $350 to $1,200 for a 10-reaction kit to more than $3,000 for 100-reaction kits. "It is always cheaper and easier to assemble a home-brew kit," says Player. "Current kits are very, very expensive."
WHERE TO BEGIN?
If you've decided to go with a commercial kit, Ginsberg's advice is first to make sure that you actually need it. "Many people that I consult with would be better off, for instance, performing a qPCR [quantitative PCR] experiment or an RNAse protection assay to answer their individual question," he says. These alternate methods are more appropriate, for example, if a researcher is investigating expression of a single or small number of genes, and they don't require RNA amplification.
If an array experiment is appropriate, Ginsberg advises researchers to consider the following issues when choosing an amplification kit: sample size and preparation, tissue and/or cell quality (RNA degrades quickly, so samples should be fresh, promptly frozen, or fixed), and especially the amount of input RNA that will likely be available. Many commercial kits will amplify reliably with large input RNA amounts, but "basically, the smaller the RNA input source, the worse the kit works."
Commercial kits recommend anywhere from one nanogram to 5 micrograms of input RNA. Some claim to work with sample sizes as small as 10 to 500 picograms, but Player has found that such claims tend not to stand up in the lab. She sets the workable lower limit for most kits at one nanogram.
Researchers also need to calculate laboratory and technical effort, cost, and goals to determine the commitment level required, Ginsberg says. "Array work is usually a long-term commitment, not a one-shot deal, as many researchers have found out, much to their dismay."
Selected RNA Amplification Kits
Despite the shortcomings of current RNA amplification kits and protocols, Player remains optimistic. Through tweaking available kits, she has almost reached her goal. "We are now down to being able to amplify 500 to 100 picograms of input material," she says. That corresponds to between 10 and 50 cells.
Future kits and protocols will likely improve by using a more efficient T7-based polymerase, priming RNA synthesis with an oligo(dT) and random hexamer, decreasing sample loss through a one-step protocol, and using a sensitive fluorescent detection signal, she says. Certain kits have already incorporated some of these improvements.
"We have come a long way in the past few years," she says, "and we are almost at the point of being able to reproducibly perform expression profiling of individual cancer cells."