Global amplification of sense RNA: a novel method to replicate and archive mRNA for gene expression analysis

Genomics. 2003 Oct;82(4):491-7. doi: 10.1016/s0888-7543(03)00115-0.

Abstract

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.

Publication types

  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Cell Line
  • DNA, Complementary / biosynthesis
  • Gene Amplification
  • Gene Expression
  • Gene Expression Profiling / methods*
  • Humans
  • RNA / biosynthesis*
  • RNA, Messenger*
  • RNA-Directed DNA Polymerase
  • Reverse Transcriptase Polymerase Chain Reaction

Substances

  • DNA, Complementary
  • RNA, Messenger
  • RNA
  • RNA-Directed DNA Polymerase