Fluorescence-based evaluation of shRNA efficacy

Anal Biochem. 2011 Oct 1;417(1):162-4. doi: 10.1016/j.ab.2011.06.008. Epub 2011 Jun 13.


RNA interference is a cellular mechanism regulating levels of mRNAs. It has been widely exploited to knock down specific protein targets. The selected interfering RNA sequence greatly influences its ability to knock down the target. Here we present a method for constructing multiple testing plasmids which express small hairpin RNAs (shRNA) targeting different regions of an mRNA. A simple fluorescence test in cultured cells allows convenient evaluation of mRNA knockdown by many different shRNAs on 96-well plates. We show that software predicted shRNAs have varying efficacies and only 2 of the 7 tested shRNAs significantly knocked down their targets.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Animals
  • Base Sequence
  • Biochemistry / methods*
  • CHO Cells
  • Cricetinae
  • Cricetulus
  • Evaluation Studies as Topic*
  • Fluorescence
  • Gene Knockdown Techniques
  • Genes, Reporter / genetics
  • Molecular Sequence Data
  • RNA, Messenger / analysis
  • RNA, Messenger / genetics
  • RNA, Small Interfering / analysis*


  • RNA, Messenger
  • RNA, Small Interfering