RNA Knockdown

RNAi has proven to be a powerful, genome-wide technique for studying loss-of-function via gene knockdown. Short interfering RNAs (siRNAs) can be transfected into cell populations to effect gene silencing and libraries of known siRNAs have been created for a wide variety of gene families. To assess the reduction in gene expression, quantitative real-time PCR is often the method of choice to measure mRNA levels. However, down-regulation of mRNA does not always correlate well with corresponding protein levels, especially those with low turnover rates.

In practice, siRNA transfection efficiencies can be low and variable, so a better approach is flow cytometric measurement of intracellular protein knockdown in situ with viable intact cells from all subpopulations. Since flow cytometry is multi-parametric, fluorescent antibodies can be used to not only assess the siRNA-targeted protein levels, but also to detect other functional cell surface marker signals in the same cell. Various subpopulations in a heterogeneous sample can be quantitatively analyzed without the use of potentially function-altering cell purification protocols.

Resources: