I am currently working with digital PCR for the detection of DNA and RNA pathogens in various pig tissues. However, I am experiencing an issue with RNA isolated from pig kidney tissue. I isolated RNA, treated it with DNase I, cleaned it using an RNA concentration cleanup column, and checked the RNA concentration and quality and it all looked good. Then proceed for the cDNA reaction and digital PCR. After PCR, I only obtained a few positive droplets (which could be only noise background) for the reference gene GAPDH and for PERV, an endogenous retrovirus in pigs. It appears that something in the kidney tissue may be inhibiting the PCR, as the same procedures on other pig tissues have yielded successful results. Has anyone encountered a similar experience or have any insights on potential inhibitors present in kidney tissue? Any thoughts or suggestions would be greatly appreciated. Thank you!
What method/kit do you use for extraction? Most kits (e.g., Qiagen, Zymo) are pretty good at removing inhibitors. Do you use positive controls for GAPDH and PERV? I would definitely check these first.
Thank you for the suggestions Péter Gyarmati. I use a QIAGEN kit for RNA isolation and a Clean-Up and Concentration column to purify the eluted RNA. Yes, I include controls for both GAPDH and PERV, and I follow the same process for ten other tissues. However, I'm only experiencing this issue with kidney samples...
Hi, there are a few additional things you can try:
- It might be worth to also test a few RT-kits (and maybe change your primer strategy too, depending on what you are using now).
- What was the cDNA input for your digital PCR reaction? Too much cDNA buffer can also inhibit PCR.
Cláudia P Muniz Interesting. I would try diluting the sample and see whether that solves anything, otherwise just try the Zymo RNA kit
Péter Gyarmati and Caroline Weydert thank you so much for the suggestions. I treated the RNA with proteinase K and then used the Zymo RNA Clean & Concentrator kit, and the ddPCR reactions worked really well. I believe that during the RNA isolation process, some enzyme from the kidney was being eluted along with the RNA, which was inhibiting the reaction.
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