What is the best method for monitoring siRNA functionality?
siRNA-mediated silencing occurs as a result of target mRNA recognition and cleavage. The status of the target mRNA level is critical for understanding the experimental system and for isolating potential issues while troubleshooting. Because of the variability in protein stability and turnover rates in biological systems, the time course and degree of protein reduction may differ significantly from that of the target mRNA. Thus, while protein measurement provides important experimental information, measurement of the silencing effect at the mRNA level is the most important and reliable indication of siRNA-mediated silencing efficiency. Once successful silencing has been confirmed at the mRNA level, subsequent measurement of target protein levels can assist in correlating mRNA reduction with phenotypic effects. Time-course studies to assess maximal protein reduction may need to be performed for protein-level studies.