I am doing qPCR to quantify silencing some of the proteins I work with. but I see that my silenced values are greater than that of the control ones.
I got higher values in silenced samples after the double delta ct analysis. Essentially, my silenced samples are showing higher transcript levels compared to the un-silenced ones.
Hi Girishkumar Kumaran
if you mean that you have higher ct, it's normal since higher ct is for less expressed genes. The higher a gene is expressed and the lower the ct will be.
all the best
fred
Hi Frederic Lepretre
I should have been more clear about what I did. I got higher values in silenced samples after the double delta ct analysis. Essentially, my silenced samples are showing higher transcript levels compared to the un-silenced ones.
OK, it means that you could have issues on your samples. is there a difference of quality between both groups?
Be sure you’re not amplifying your own dsRNA. Are your qPCR primer designed outside the region targeted by your dsRNA ?
Frederic Lepretre I think cDNA quality isnt a problem. As Andrea Becchimanzi suggested, I used primers for my own dsRNA. I have ordered new primers outside the dsRNA region and once I complete the new round of qPCRs I will update all of you. Thank you very much for you all of your comments and suggestions. I highly appreciate it.
Hi Girishkumar Kumaran
cDNA won't present issues, you're right, but the problem is to produce this molecule in good conditions and in a proper way. degraded RNA show lower length RNA and introduce a bias in representation of all expressed genes from the cells. as all typical molecular biology techniques, issues are often in the original samples and therefore all samples must be at same shape.
all the best
fred
Hi Girishkumar Kumaran
Have you checked the efficiency of your reactions? If the silenced sample have higher amplification efficiency that would cause higher ddCt.
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