So until yesterday my qPCR was working fine. Today, when i started with my qPCR run i found very low ct values or flat lines.
The Ct values until yesterday were between 24-30 in my treated sample and in my control sample it was 32-34.
Today i ran the same samples but as said above the ct values were between 4-8 or undetermined and the same thing happened with my control sample? What are your opinions on what went wrong? To double check this i repeated this twice and both the time i got similar kind of results.
But i extracted RNA from fresh brains from the same animal and took the left hippocampi (i had used the right hippocampi before) made cDNA and then ran them again (3rd qPCR). Surprisingly, I got Ct values like before and also some old samples (which i analyzed again with this run) did show ct values.
Have the primers gone bad? (I prepared fresh stock on Tuesday)
something wrong with the cDNA? (have aliquots and aliquots doesn't last for more than 2 runs)
Something wrong with the SYBR Mastermix? (Thermo scientific)
Kindly advice and please give your opinions on what went wrong.