Optimization of siRNA experiments - (Sep/18/2008 )

Hi,

I am starting a experiment where i wish to use siRNA to target a gene in MCF-7 cells.

I have a pre-validated siRNA and a negative scrambled control.

I began my optimization by using a range of siRNA:transfection reagent ratios and anaylzed the gene expression using semi-quantitative RT-PCR. However, i have now noticed that alot of people seem to use real-time and i was wondering if my optimization results will be accurate enough to allow me to proceed with the next stages of the experiment or should i do a real-time pcr to confirm?

Also i was just wondering if its necessary to have a positive control as i don't want to do all the work & then be told that i should have had one!

Many thanks in advance!

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make two-fold serial dilution of all RNAs (both from control and siRNA treated sample) in RNAase free water till 10-12 dilutions
and using these diluted RNAs as template perform RT-PCR and do it in duplicate or triplicate.

the reason is that control samples will have high copy of homologus RNA(against which you are doing siRNA experiment) as compared to other siRNA treated samples. then putting semiquantitative RT-PCR will tell you up to what dilutions u are getting amplification. for example if u are getting amplification till 10th dilutions of control sample, then siRNA treated samples will give amplification in less than 10th dilutions (if gene silensing is sucessful). another advantage of this is that uou can estimate the RNA levels in both the samples (that is also quantitative) using spectrophotometer

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