Griffin:siRNA transfection: Difference between revisions

'''Mismatch or scrambled siRNAs:''' These are often of somewhat limited value. A scrambled sequence is too unrelated to the 'active' probe to function as a truly informative control. Occasionally, a one- or two-base-pair change in the middle of the antisense can be a useful negative control if the siRNA effect is clearly ablated. However, such a change may have unanticipated effects by converting an siRNA into a miRNA (micro RNA) that inhibits translation through a pathway closely related to siRNA.

'''Basic controls:''' siRNAs exert their effects through a growing number of surprisingly diverse mechanisms in addition to selective degradation of the targeted mRNA, such as specific effects at the chromatin level. Currently, siRNAs, unlike long double-stranded RNA (dsRNA), are not thought to trigger general translational attenuation through the interferon response. However, this also remains a hotly debated possibility for some short siRNAs, or at least mixed populations retaining some longer dsRNA species. In addition, the closely related miRNAs do function through target-specific translational attenuation. Thus, it is important to show reduction of expression at the mRNA and protein level, as well as a functional readout where available. If both message and protein are ablated, the response is 'classical' RNAi. In contrast, if only the protein is reduced, the chances are that a miRNA-related translational mechanism is at work. It should be noted that although this set of controls should be regarded as best practice, the functional control listed below renders analysis of the mRNA non-essential. Additional useful controls are available for unintentional activation of global translational repression through the interferon response (commercial assay kits or expression of unrelated proteins). Although centromeric or other emergent chromatin effects are harder to generate controls for, global gene expression may function as a control for any non-specific effects on gene expression.

'''Quantitative controls:''' Titration of the siRNA is strongly encouraged. RNAi is often extremely effective already at minimal concentrations, and titration to the lowest possible levels reduces the chance of side effects as well as providing a graded readout of the effect. This is especially important because the RNAi machinery (the RISC complex in particular) is saturable, at least in some settings. Again, the rescue control outlined below is especially important when high levels of siRNA must be used.  Protein levels should be assessed with quantitative techniques, such as quantitative western blotting, to allow for an accurate estimate of the level of reduction.

'''Functional controls:''' The ultimate control for any RNAi experiment remains rescue by expression of the target-gene in a form refractory to siRNA (ideally within the physiological range). This can often be achieved by utilizing one or more silent third-codon point mutations within the targeted region, although controls for fortuitous miRNA effects are desirable. Translational effects can be avoided by utilizing siRNAs targeted against 3'-untranslated regions (UTRs), which are non-essential for rescue expression from a plasmid.  The use of recently developed vector-based RNAi systems will alleviate some of the technical hurdles of rescue expression.

'''Multiplicity controls:''' a good way to enhance confidence in RNAi data is to demonstrate a similar effect with two or more siRNAs targeted to different sites in the message under study. Alternatively, the RNAi approach is usefully supplemented by alternative methods, such as those described above.