I used the same cDNA and primer to perform qPCR twice. However, the Ct value varied about 10 cycles(25 vs. 35). What's the reason? gDNA wipeout buffer is used.
Did you perform duplicates? Are the values the same in both sets of duplicates? Either the DNA has degraded over time or there could be a pipetting error, for example if you didn't correctly mix enough DNA into the reaction etc.
As indicated in previous answers that magnitude of difference points to something being seriously flawed
I would re make your cDNA; aliquot into 20ul aliquots and only try to thaw each aliquot 1- 2x and then discard: cDNA withour purification is actually unstable when subjected to freeze thawing. You can render cDNA more stable by adding 0.5mM EDTA or column purifying but the simplest and most often advocated solution is to make fresh. I have a rule never to keep cDNA for quantitative work at -20C for more than 1 month even without freeze thawing
https://www.researchgate.net/post/Can_we_purify_cDNA_using_spin_columns
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