Single-cell cDNA-PCR: removal of contaminating genomic DNA from total RNA using immobilized DNase I

Biotechniques. 1992 Nov;13(5):726-9.

Abstract

A procedure utilizing immobilized DNase I that allows the efficient amplification of cDNA by PCR from a single cell in the absence of contaminating genomic DNA is described. DNase I treated, total RNA derived from single cells was reverse transcribed into cDNA followed by PCR using beta-actin and c-fos specific primers that recognize different exons of the respective genes. Amplification products corresponding to cDNA, but not to genomic sequences, were detected after treatment with immobilized DNase I in samples previously shown to be contaminated with genomic DNA. This method allows the efficient removal of DNA contaminating total RNA derived from a single cell.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Artifacts
  • Base Sequence
  • DNA / analysis*
  • Deoxyribonuclease I*
  • Enzymes, Immobilized
  • Molecular Sequence Data
  • Polymerase Chain Reaction / methods*
  • RNA, Messenger / analysis*

Substances

  • Enzymes, Immobilized
  • RNA, Messenger
  • DNA
  • Deoxyribonuclease I