Amplification of RNA transcripts using terminal continuation

Lab Invest. 2004 Jan;84(1):131-7. doi: 10.1038/labinvest.3700005.

Abstract

A new methodology has been developed to amplify RNA from minute amounts of starting material. Specifically, an efficient means of second-strand (ss) cDNA synthesis using a sequence-specific 'terminal continuation' (TC) method is demonstrated. An RNA synthesis promoter is attached to the 3' and/or 5' region of cDNA utilizing the TC mechanism. The orientation of amplified RNAs is 'antisense' or a novel 'sense' orientation. TC RNA amplification is utilized for many downstream applications including gene expression profiling, cDNA microarray analysis, and cDNA library/subtraction library construction. Synthesized sense TC-amplified RNA can also be used as a template for in vitro protein translations and downstream proteomic applications. The TC RNA amplification methodology offers high sensitivity, flexibility, and throughput capabilities. A likely mechanism is that the TC primer binds preferentially to GC-rich CpG islands flanking 5' regions of DNA that contain promoter sequences. Following TC RNA amplification, a large proportion of genes can be assessed quantitatively as evidenced by bioanalysis and cDNA microarray analysis in mouse and human postmortem brain tissues.

Publication types

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

MeSH terms

  • Animals
  • Brain / cytology
  • Brain / physiology
  • Brain Chemistry
  • DNA Primers / chemistry
  • DNA, Complementary / analysis*
  • DNA, Complementary / genetics
  • Gene Expression Profiling
  • Humans
  • Mice
  • Mice, Inbred C57BL
  • Nucleic Acid Amplification Techniques / methods*
  • Oligonucleotide Array Sequence Analysis / methods*
  • RNA / analysis*
  • RNA / genetics
  • Sensitivity and Specificity

Substances

  • DNA Primers
  • DNA, Complementary
  • RNA