Global amplification of sense RNA: a novel method to replicate and archive mRNA for gene expression analysis.
We have developed a procedure to amplify mRNA into sense RNA (sRNA) so as to create a regenerating biorepository representing the complex mRNA profile in the original sample. The procedure exploits the template-switching activity of reverse transcriptase to incorporate RNA polymerase binding sites upstream of single-stranded cDNA (ss cDNA). Limited PCR was used for double-stranded DNA (dsDNA) synthesis. sRNA was synthesized from PCR products by in vitro transcription (IVT). sRNA was evaluated by real-time reverse transcription (RT)-PCR. sRNA synthesis was successful with RNA from human cell lines and tissues, yielding 2000- to 2500-fold amplification of glyceraldeyde-3 phosphate dehydrogenase (G3PDH). The size of sRNA ranged from 3.0 to 0.1 kb. sRNA synthesis preserved the relative differences in plant mRNAs spiked at abundance ranging over 5 orders of magnitude (0.00001-0.1%). This reflects the high fidelity of sRNA synthesis for mRNA as low as 0.3 copies/cell. sRNA is amplified synthetic mRNA in the 5'-->3' direction; the appropriate template for any gene expression analysis.
D002460:Cell Line; D018076:DNA, Complementary / Q000096:biosynthesis; D005784:Gene Amplification; D015870:Gene Expression; D020869:Gene Expression Profiling / Q000379:methods*; D006801:Humans; D012313:RNA / Q000096:biosynthesis*; D012333:RNA, Messenger; D012194:RNA-Directed DNA Polymerase; D020133:Reverse Transcriptase Polymerase Chain Reaction
D018076:DNA, Complementary; D012333:RNA, Messenger; D012313:RNA; D012194:RNA-Directed DNA Polymerase