Determination of Interferon-Pathway–Related Gene Induction during RNAi

Experimental approaches using short interfering RNA (siRNA) molecules to specifically silence gene expression have become more widely used in recent years. As for all techniques in molecular and cellular biology, the importance of protocol optimization and the use of appropriate controls cannot be overestimated.

While the induction of interferon-pathway–related stimulatory processes during RNAi is usually attributed to long, double-stranded nucleic acids, it can also be a concern during transfection of short siRNA molecules or in other RNAi-related experiments.

A number of parameters, including siRNA chemistry, transfection reagents, cell culture conditions, and siRNA concentration have the potential to cause interferon-pathway–related responses. These responses could interfere with the specific biological effect caused by the siRNA-mediated knockdown of the gene of interest, causing misleading results.

Here we describe assays for quantification of mRNAs which are derived from induced transcription of well characterized interferon-pathway–related genes, using quantitative, real-time RT-PCR.

The assays are specific for STAT1, IL6, IFNa1, IFNß1, IFIT1, IFITM1, OAS1, or OAS2. We performed the assays after cells were transfected with functional amounts of synthetic siRNAs or with polyinosinic-polycytidylic acid (poly(I)poly(C)), which is a long, double-stranded RNA that induces the interferon pathway. Although poly(I)poly(C) transfection caused dramatic induction of several interferon pathway genes in these cells, as indicated by the assays, such induction was not seen when siRNAs from QIAGEN were transfected.

We conclude that these assays are valuable tools for optimization of experimental conditions and for use as controls, ensuring successful RNAi experiments.