siRNAs exert their effects at the mRNA level. Therefore, the preferred assay for siRNA validation and transfection optimization purposes is one that monitors target mRNA levels.

The simplest and arguably the best assay for siRNA validation and transfection optimization relies on qRT-PCR to measure target transcript levels in gene-specific siRNA treated cells versus negative control siRNA treated cells. Applied Biosystems TaqMan Gene Expression Assays, available for >41,000 human, mouse, and rat genes, are ideal for this purpose.

Ambion's siRNA database provides links to individual Taqman Gene Expression Assays matched to gene-specific Silencer Pre-designed and Validated siRNAs, which makes finding both siRNAs and real-time PCR assays to your gene of interest a snap.

Although it is necessary to monitor mRNA levels to validate siRNAs, most researchers wish to determine the extent of knockdown at the protein level. Ambion's PARIS Kit (PARIS stands for Protein and RNA Isolation System) provides a simple method for isolating total RNA and protein from the same sample. RNA isolated with the kit can be used for RT-PCR, array analysis, Northern blotting, or other analytical technique, and the protein lysate is compatible with Western blotting and, since native protein is recovered in most cases, enzymatic assay.

The most careful of researchers will wish to correlate siRNA, target mRNA, and target protein levels. Isolation and detection of small RNA, however, requires modified, and in some cases, completely different techniques than isolation and detection of longer RNAs. The new mirVana PARIS Kit is like the PARIS Kit in that it isolates both RNA and protein from the same sample, but it also isolates small RNA species, including siRNAs. The mirVana miRNA Detection Kit provides a way to quantitate siRNA levels in cell populations, and can also be used to examine target mRNA levels.