Thank you in advance for any help.
I have been running qRT-PCR's for years using SYBR green and the STEPONE PLUS system. But recently, I am having this problem that I cannot seem to come up with an excuse for.
I was running qPCR validation on hits from some next gen. seq. data using 4 cDNA samples for about two weeks, looking at different genes that were shown to be upregulated for our condition.
All of a sudden, one day, I was started getting no amplification with multiple primer sets, so of course I figured all I ran into some bad primer sets. Moved onto the next set of primers, and still no amplification. I always test my primers beforehand, and this many primer failures has never happened to me before, so I went back and used the same stocks of cDNA that I had been, and primer sets that had originally worked in the first few days that I was doing this, and now it says no amplification.
Made new diluted cDNA stocks-- primer sets that once worked, still won't work.
I tested a different set of cDNA (completely un-related to this) and primers that worked for it once, and the qPCR worked.
So, now I am thinking it is my cDNA. But, I don't understand how it worked for weeks, and now I cannot get any amplification (not even gapdh), when it once worked so well. (Not a freeze thaw issue)
Anyone ever have this problem? Or can think of ANYTHING?