I'm performing qPCR with Roche SYBR green Fast Start (Before I used Bio-Rad and everything worked).
I added disocciation curve in tem 52C, my primers melt at 54C, my reference gene in 57C.
My reference gene showed nice disocciation curve peak, BUT NOT any of my genes! No curve at alll of them (cDNA, no-RT, NTC). The Ct value of amplification looks very nice for cDNA, for NTC there is no amplification... just this lack of melting curves seems very strange. What could possibly went wrong?