Evaluation of small hairpin RNA silencing efficiency in live cells by cotransfection of two fluorescent probes

During recent years, the selected knockdown of protein expression by RNA interference has received rapidly growing interest. Although short interfering RNA (siRNA) target designers apply strict selection parameters, the deduced small RNAs need to be tested for their silencing potency. Here we describe a fast and efficient method for evaluating the silencing efficiency of target gene by small hairpin RNAs (shRNAs) in mammalian cells. Cells were cotransfected with two vectors: one containing shRNAs as well as the coding region for green fluorescent protein and one containing a chimeric fusion construct encoding red fluorescent protein coupled to the synaptic vesicle protein SV31. The efficiency of various shRNAs was directly monitored in vivo by fluorescence microscopy.