Despite satisfactory qPCR curves for the positive control and calibrator, I observe a lack of signal from the VIC dye (reference gene) in my experimental samples after bisulfite conversion. I have tested multiple conversion kits and confirmed the post-extraction DNA concentration. What could be the potential causes of this issue, and what strategies would you recommend to resolve it?
Please note that we have repeated the technique several times with the following modifications:
1- Change of DNA extraction kit
2- Change of bisulfite chemical treatment kit
3- Change of positioning of controls and samples in the qPCR tubes
Note: We measured the DNA concentration after each extraction and after bisulfite treatment.
What do you use as the reference gene? The same amplicon (with methylation independent primers), but with a different probe? Or a totally different gene?
Have you considered bisulfite conversion free qPCR/dPCR? It might be another option.
Basically, you make sure that your target amplicon contains sites that are recognized by a Methylation Sensitive Restriction Enzyme. More info can be found in this publication for qPCR: Chapter Multiplexed and Sensitive DNA Methylation Testing Using Meth...
and in this one for ddPCR:Article Quantification of DNA methylation independent of sodium bisu...
Hello,
Thank you so much for your answer.
We’re using a qpcr kit for the detection of the methylation of the gene MGMT for patients with Glioblastoma ( the name of the reference gene is not mentioned)
Please note that we’ve already used this technique a few months ago and it worked but now it didn’t ( we used one of the previous samples and it still didn’t work this time) we’re doubting the bisulfite conversion kit so i have a question please : it’s indicated in the protocol of our qpcr kit « Bisulfite treatment of the isolated DNA using the EZ DNA Methylation-Lightning™️ Kit (Zymo Research, Cat#D5030) » Does that indicate that the technique won’t work if we use a bisulfite conversion kit other than the one provided by Zymo Research??
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