I’m doing RT-PCR experiment using Quercetin treated Hepa1c1c7 cells. i want to see the gene expression of HO-1 gene. The first time it was all good, but I couldn’t replicate the exper and ended up failing all the time. the electrophoresis result always shows only beta actin band and not HO-1. I’ve been trying so many different thing such as increasing the cycle, change the order of my pcr buffer mixture, diluting the cDNA, etc. I’m thinking if i made a mistake during RNA extraction or reverse transcription, both of the bands should not be visible, but beta actin is there. And i made the pcr buffer in one tube, then i divided into two, then i added the primer mix. I always made sure i took the right amount of primer and that it was mix well, same with biotaq. I really don’t know what did i do wrong, please help.
You can try the following in your future tests;
Sample re-extraction and ethanol percipitation
Using new RNA extraction kit and cDNA synthesis kit from other companies
Using new master mix
Change your denaturation time, annealing time, and extension time
Also, suitable concentration for your cDNA is 100 ng per 20 µL reaction, and suitable concentration for your primers is 0.2 µM.
There is one thing you could try, which could not be absolutely the reason, but it is worth trying. As we store our cDNA in tubes which are not low DNA binding and this could affect result, so before PCR reaction thaw the tubes at 37 C for 30 minutes and use the cDNA for your RT PCR. I got very consistent results.
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