I performed a global methylation test (ALU and LINE) with qMSP method, using TaqMan and TaqMan Universal Master mix probes (reference gene ACTB) in buffy coats. The conversion was performed using Methylation Gold Kit (Zymo Research).
The problem is the non-repeatability of Ct results - both for the ALU and LINE genes. However, for ACTB, the obtained Ct values are above 33 cycles or 'undetermined'.
Are there any discrepancies between repetitions of one sample above 1-2 cycles? How can I optimize the reaction so that the obtained Ct for ACTB is no greater than 30 (tests with different concentrations of primers and probes did not bring any significant improvement)?
Can I ask for help in solving this problem?
Assuming your efficiency is 95-105%, then a Ct value above 30 means the molecule of interest is just very low-copy.
You can try adding in more template volume to start with more molecules.
Good luck!
This is something we have already tried, however we did not get the expected improvement in the results. Nevertheless we will continue to try. Thank you very much for your suggestion.
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