How can researchers amplify minute volumes of RNA to produce enough aRNA for an array but avoid getting an amplification bias?
For a while, several leading researchers have said linear amplification is the answer. With PCR, the amplification follows a sigmoidal curve, starting out horizontal, then becoming linear, and tapering off towards the end. Amplification that is not stopped at the linear phase of the cycle will skew along with this curve. But with linear amplification, there is no curve.
Until now, however, the only commonly available form of linear amplification, has been the Eberwine T7 protocol, which many researchers have found to be less reliable than PCR and to require extensive fine tuning, according to Rajiv Raja, a scientist at Arcturus, of Mountain View, Calif..
Arcturus has recently introduced a commercial alternative to this protocol, a linear amplification kit called RiboAmp. The kit includes amplification enzymes and reagents, DNA and RNA purification reagents, and DNA/RNA purification columns. It can be used for ten amplification reactions and costs $1,125.
The basic premise is the same as the T7 Eberwine protocol, except we have optimized and troubleshot each and every aspect of the process, said Raja. During our development, we have done 1,500 to 2,000 reactions. It is very robust and reproducible, offering over 99 percent success.
In the T7 protocol, RNA is first isolated from cells and T7 promoter sequences are attached to the end, according to Mark Schena, who described the process in the first chapter of Microarray Biochip Technology (Eaton Publishing, 2000). This cDNA is then transcribed in vitro using a T7 polymerase, yielding a larger amount of aRNA, typically 100 fold. The aRNA is then labeled and a reverse transcriptase is added to transcribe it back into cDNA, which can be hybridized to an array.
Schena suggested in Microarray Biochip Technology that the microarray field adopt this strategy as the method of choice when mRNA amplification is required.
Other advantages of this process, in addition to avoiding the problem of skewed aRNA populations, also include the fact that the reverse transcriptase added to the aRNA after amplification does a better job of incorporating the fluorescent dyes into the transcripts than DNA polymerases, Schena wrote.
Raja also pointed out that the method does not require any primers specially designed for every gene, instead allowing linear amplification of the whole genome at a time. You can get a whole genome assay from as little as one nanogram of total RNA, he said.
Recently, some groups, including Wilhelm Ansorges lab at the European Molecular Biology Laboratory and a number of core facilities (see p. 7), have been heeding Schenas suggestion. Others have been discussing this protocol on the Gene-Arrays listserv, and have indicated they are trying it.
The protocol does, however, offer some disadvantages. It does not always amplify RNAs that have complex secondary structure, potentially eliminating between two and five percent of the transcriptome, said Raja.
But this disadvantage does not seem to be driving away researchers who are looking for a way to reliably amplify small volumes of RNA, especially those with clinical samples. Arcturus, which initially beta tested the kit in 20 labs around the world, has been receiving a steady flow of orders since it first introduced the RiboAmp kit at a National Institutes of Health microdissection conference in August, Raja said. Most of the time I am on the phone now answering questions from customers.