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RNA Arbitrarily Primed PCR 245
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Microarray Analysis Using RNA
Arbitrarily Primed PCR
Steven Ringquist, Gaelle Rondeau, Rosa-Ana Risques,
Takuya Higashiyama, Yi-Peng Wang, Steffen Porwollik,
David Boyle, Michael McClelland, and John Welsh
1. Introduction
RNA arbitrarily primed polymerase chain reaction (RAP-PCR) has been used
extensively to identify differentially regulated genes (1–6). The RAP-PCR method
begins with conversion of RNA into cDNA, followed by arbitrarily primed PCR.
The technique uses arbitrarily primed PCR (7–11) to amplify cDNA stretches lying
between sequences that, by chance, match arbitrarily chosen oligonucleotide primers
well enough to initiate primer extension. In earlier applications of the method, the
complex mixture of products was resolved by polyacrylamide gel electrophoresis
(PAGE), yielding highly reproducible fingerprints characteristic of the RNA source.
Differences between fingerprints resulting from differentially expressed genes were
verified by Northern blot analysis or reverse transcription (RT)-PCR.
Here, a combination of RAP-PCR and cDNA array technology is described (see
Note 1, refs. 12–14), which provide dramatic improvement in detection sensitivity and
ease of analysis when compared with PAGE. Typically, PAGE analysis of a RAP-PCR
examines ~50 to 100 genes, whereas microarray analysis of RAP-PCRs examines
~10 to 20% of the genes represented on the microarray, simultaneously. With PAGE,
characterization of differentially regulated genes requires laborious purification of the
band, cloning, and sequencing (15). With microarrays, the identity of the gene is known
immediately because the arrayed sequences are known. As is generally understood with
microarrays, the microarray-based RAP-PCR approach can quickly narrow all mRNAs
down to a subset that exhibits regulation under the conditions examined. However, the
RAP-PCR method selectively examines a different population of mRNAs than does
probe from mRNA or total RNA: RAP-PCR selectively samples the complex, rare
class of mRNA, presumably as the result of the greater likelihood of the arbitrary
primers encountering a sufficiently good match in the complex class to enable primer
extension. This enables the method to measure changes in RNA abundances for rare
transcripts with greater ease than with simple oligo (dT) n priming of mRNA for probe
synthesis. Iteration of the method using different arbitrary primers allows greater
coverage of the mRNA population to be achieved.
From: Methods in Molecular Biology, Vol. 226: PCR Protocols, Second Edition
Edited by: J. M. S. Bartlett and D. Stirling © Humana Press Inc., Totowa, NJ
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