qPCR: band in gel, but super high Cq? - (Nov/12/2012 )
sorry for the late answer, but was on some holidays for an extended weekend.
Here are my results with this particular PCR.
cDNA 1:2.5: Cq ~35
cDNA 1:10: Cq ~37-39
Gel: right band at 132bp
cDNA 1:2.5: Cq ~32
cDNA 1:10: Cq ~28
Gel: primer dimers appear
Product not the right size (200bp instead of 132bp)
<*>Different size for 1:10 and 1:2.5!!
Melting curve: Primer dimers visible!
Slightly different Tm for 1:10 and 1:2.5 samples
All in all it seems like the whole Primer plus Probe combination is messed up. Dimers. different band sizes?! Got new primers today and will test them.
At least I know the gene is there in some way. Tried optimising this PCR in a different tissue where we weren't sure about expression levels. At least I know now it was the assay itself.