Hello!
I am optimizing conditions of stem-loop RT-PCR to compare the expression of miRNAs in different cell lines.
Now I'm performing the reaction in one step: RT with stem-loop primer and subsequent real-time PCR with a pair of primers and SYBR Green - all in one reaction. So my reaction mix contains three types of primers, heat-inactivated reverse transcriptase, hot-start Taq polymerase, SYBR and all the nessesary components such as ddNTPs and Mg2+.
I use synthetic miRNA and total RNA from human cells as matrices.
When working with synthetic miRNA, everything goes well (Figures 1 and 2), amplification plot is ok and there is a single peak in the melting curve.
But with total RNA I observe a decline of fluorescence in the plateu phase of amplification (Figure 3) and 2 product peak in the melting curve (Figure 4).
Reference gene amplification plot doesn't have such issues.
What are the reasons of the decline of fluorescence after the 30th cycle?
What are the possible variants of the second product here? Primer-dimers observed in no-template control have Tm = 80 degrees C.