Hello! I've encountered some challenges with Traditional PCR. I've successfully conducted RNA extraction, quantification, and integrity checks, all yielding positive results. (first image its from the integrity of the RNA)
Moving forward, I proceeded with RT-PCR, followed by PCR endpoint analysis using Actin primers. My experimental design involves four treatments, including Ctrl, Resveratrol, LPS, Resveratrol+LPS, with two samples for each treatment.
However, I've encountered an issue where only the controls are being amplified during the PCR endpoint, despite using the same mix for all samples in both the RT-PCR and Traditional PCR for Actin. I'm puzzled and unable to pinpoint the source of this discrepancy. Any insights or suggestions would be greatly appreciated.
The second image its the results of the PCR.
Hi,
In my opinion, RNA damage might be the problem.
Besides, after the total RNA extraction step, did you go directly with the reverse transcription step to reverse RNA into DNA and process PCR? Normally, I will process reverse transcription as quickly as possible or keep the RNA samples at -80 degrees until use. I also avoid free thawing, which may break RNA.
I hope this information may help.
Best,
Tien
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