Fluorescence-based evaluation of shRNA efficacy

Abstract

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.

Figure 1

Reporter construct and shRNA construct. (A) A CMV promoter drives expression of a single transcript containing the GFPns coding sequence followed by a piece of the target cDNA sequence and a polyA stop signal. The cDNA sequence acts as a 3′UTR and only GFPns protein is synthesized. (B) A tetracycline-inducible promoter (Ptet) drives the expression of the RFP-shRNA transcript with the shRNA acting as the 3′UTR. Different double stranded shRNA sequences with compatible ends, made by annealing pairs of oligonucleotides, can be readily ligated into the two BspQ1 restriction sites between the 5′mir30 and 3′mir30 regions. The Ubiquitin C driven reverse transactivator (rtTA) is also in the plasmid, which is necessary for inducible expression.

Figure 2

Knockdown of the specific mRNA targets by shRNA expression. CHO cells were cotransfected with a reporter construct expressing GFPns-D2L fusion transcript and tRFP-shRNA constructs targeting D2L. Panels A–C shows the same field of cells in a well without doxycycline induction viewing through a green filter, a red filter, or a merged view of the two. Panels D–F are similar but showing cells in a well with doxycycline induction. The presence of doxycycline induced strong tRFP-shRNA expression (Panel E versus B) paralleled by substantially lower GFP-D2L expression (Panel D versus A). Panel G shows results of quantitation using ImageJ analysis of fluorescence expression. Each bar represents the average GFP expression of 6 wells presented as the ratio of GFP expression to that in the uninduced control wells. Only sequences A and D were able to reduce GFP expression significantly (p<0.05) compared to the control wells.