The SMARTpool reagent does not appear to silence. What could be wrong?
In most cases, when a SMARTpool reagent does not silence as expected (> 75% reduction of the target mRNA), it is due to inefficient delivery of siRNA into the cells. We recommend optimizing siRNA delivery conditions using a positive control siRNA. If transfection efficiency is determined to be optimal using the appropriate controls, several additional experimental factors should be considered, such as siRNA concentration, the detection method, and the time point of detection.
Stock concentrations of siRNA are typically between 20 uM and 100 uM and can be verified spectrophotometrically by measuring the absorbance at 260 nm. Once the stock concentration has been confirmed, siRNA concentrations used in transfection experiments should be reassessed to ensure that they are at the desired concentration (i.e., between 1 and 100 nM).
Detection at the mRNA level:
Cleavage of a target mRNA sequence by siRNA is an RNA-mediated event and thus mRNA level detection is the best method to evaluate functionality of siRNA-based reagents. While mRNA levels are often decreased within 24 hours post-transfection, there are instances where maximal reduction may be observed at 48 hours or more, depending on the target. Factors that can influence the time course of mRNA level reduction include gene expression levels (whether endogenous or exogenous) under the control of strong promoters and mRNA turn-over rates.
Detection at the protein level:
When assessing protein levels, it is imperative to evaluate the specificity of the antibody for the particular gene product as well as conduct time course studies ranging from 48 to 96 hours or more, depending on the protein of interest. We highly recommend verifying observations at the protein level with mRNA level detection methods. If the problem persists, we encourage the investigator to contact Dharmacon Technical Support (Ts.firstname.lastname@example.org).