We obtained change in iNOS expression through Reverse trascriptase PCR, but failed to get change in fold expression through real time PCR. The CT value obtained was quite High 32-33 and almost same with the control. Quality of cDNA was good because we obtained the desired result with other cytokines. Is it possible to get fold change with iNOS by real time PCR.
Thanks
Hi, I'm sorry because of my expertise in physiology and I can not help you
At the basal levels (unstimulated) there should be no mRNA for iNOS so you that is why you get high Ct for your qPCR. Try stimulating cells with a know stimulus that leads to transcription of iNOS, thus instead expressing your data as fold change from basal, you can express it as fold change from stimulus X.
However, if you are not detecting any iNOS mRNA using qPCR, but you could detect it on the same sample using conventional PCR (and sequencing confirmed that the band is of iNOS), then you would need to change primers for qPCR.
Hope this helps
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