we are setting up a new protocol for a RT-qPCR, for this purpose we are looking for the best control gene for our experiments.
The curves for the control genes are very good, but those for the transcript to be measured are strange.
We are trying to use primer pairs for 3 control genes:
The template is a cDNA.
Starting from the same sample of RNA, we synthesized the cDNA using 3 primer mixes:
Mix A - gene-R + CTCF-R
Mix B - gene-R + PRKC-R
Mix C - gene-R + U6-R
the primer "gene-R" contains a tag, meaning that we have about 20 nucleotides complementary to the gene transcript and about other 20 nts that are unique and can anneal to another oligo whose sequence is not present on the genomic DNA ("tag").
The qPCR mixes are prepared like these:
Mix A - CTCF-F + CTCF-R
Mix B - PRKC-F + PRKC-R
Mix C - U6-F + U6-R
Gene in analysis
Mix A - gene-F + tag
Mix B - gene-F + tag
Mix C - gene-F + tag
While the curves for all the control genes and that for "Mix C - gene-F + tag" are very good (not shown), those for Mix A and B look strange (shown in the attached picture), especially Mix A shows some sort of "bump".
What does it mean? Does anybody already experienced Real times with this strange shape? Aspecifics? Dimers? How do I get rid of it?