Urgent Help needed: RNA-Interference, rt-pcr and Western-Blot do not match - (Feb/03/2010 )
I have the following problem. I have used 6 different shRNAs to achieve a gene-knockdown in ZR-75-1 breast cancer cells. Only one shRNA did work. The Western-Blot Analysis showed that one shRNA was successful and that the remaining expression of my Protein is about 1/8 of the wildtype expression.
Unfortunately when I try to proof these results by quantitative realtime pcr, the expression level of the targeted Protein is as high as in the other samples, in which no knockdown could be observed by western-blotting.
How can that be?
RT-PCR conditions have already been optimised... Primers are working. Samples have not been mixed up.
Could it be possible that the shRNA 3 is not fully matching the Sequence of the target mRNA and therefore does not lead to a degradation of the mRNA but instead to a storing of the mRNA in p-bodies? This would explain why the mRNA could still be detected in RT-PCR...
I need your help guys
What do u think?
Ok i did a search on the shRNA and it is supposed to match 100% in the orf of the mRNA...
Maybe you have a feedback loop? Cells detect a decrease in mRNA, so they ramp up transcription of the gene? Thus you don't detect a difference in mRNA but you do in protein because the mRNA is being degraded. This is just a hairbrained idea...but if you thought it halfway decent you could check it by doing Chromatin Immunoprecipitation for Pol II at the promoter region of your gene following treatment with shRNA. Would have increased binding of Pol II following shRNA treatment.
Just a crazy thought, that's what you wanted right?