I was working on a project that involved performing quantitative RT-PCR (qPCR) from cells obtained by laser capture microdissection. Without amplification, I was unable to detect most of my targets. I tried using an RNA amplification kit but found that one round of amplification was not adequate. Because the procedure was time-consuming and expensive, I did not want to perform multiple rounds of amplification to prepare the samples. Moreover, reviewers may question studies involving RNA that has been amplified several times. At that point, I tried the TaqManŽ PreAmp Master Mix, which involves preamplification of the cDNA rather than the RNA. Although the product was initially only available together with the quantitative PCR master mix, it can now be purchased alone. While the price of the reagent is somewhat high, it probably is not more expensive overall than RNA amplification.
The procedure involves performing a 10 cycle or 14 cycle PCR reaction using a pooled mixture of your qPCR assays as primers. The 10 cycle protocol is suitable when only a small number of TaqManŽ assays are being analyzed and has the advantage of a shorter PCR time. On the other hand, the 14 cycle protocol can generate enough starting material for up to 200 reactions. The amount of hands-on labor is minimal, since most of the added step is a PCR reaction. The preamplification PCR is performed in a standard thermocycler so there is no need to tie up the real-time instrument. Up to 100 gene expression assays can be pooled. Although the reagent is designed for use with TaqManŽ assays, I also used it successfully with the Universal Probe Library from Roche. You perform a standard reverse transcription reaction using unamplified RNA and then use the cDNA together with the PreAmp Master Mix and the pooled gene expression assays. The reaction is designed for use with 1-250 ng cDNA. Although Applied Biosystems recommends using their cDNA synthesis reagents, I obtained excellent results using a product from another company.
One potential concern is that the various amplicons might not be amplified uniformly without bias. This issue arises for all RNA and cDNA amplification procedures. Applied Biosystems provides a protocol for assessing the preamplification uniformity using a sample that is not limited. The master mix has been validated with partially degraded samples as well, making it suitable for use with postmortem tissue and formalin-fixed tissue. My own work involved postmortem brain tissue and it gave excellent results.
One disadvantage that I noticed was that because the RNA is not amplified before adding it to the reverse transcription reaction, it may not be possible to measure the quantity and quality of the RNA in each sample. This issue makes it difficult to normalize the RNA input for the reverse transcriptions and to perform adequate quality control testing.
I have recently switched labs and my new lab is preparing to perform laser microdissection studies. I have recommended that we switch from the RNA amplification procedure used in the past to the TaqMan PreAmpŽ Master Mix.
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Tara L. Lauriat
Postdoctoral Fellow
Program in Structural and Molecular Neuroscience
McLean Hospital
Unspecified
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