Hi, has anyone experience of miRNA qPCR panels, especially QIAgen miRCURY LNA miRNA PCR panel? I performed a qPCR run of 384 well plate (panel I) from SW480 cell line according to the procedure, but got very high Ct values. Spike-in control of RNA extraction, UniSp2, amplified at cycle 31.7, and Sp4 and Sp5 didn't amplify at all.
The total RNA yield was good (about 1 ug / ul). For the next run, I should probably add more RNA to cDNA synthesis and further to qPCR run, but how much?
I did a quick calculation:
Concentration differences between Sp2, Sp4 and Sp5 are 100-fold. Thus Sp4 should amplify 6.6 cycles later that Sp2, and Sp5 6.6. cycles later than Sp4. If I add cDNA template 10-fold more (increasing the amount from 20 ng to 200 ng), all Sp-controls should amplify 3.3 cycles earlier than in first run. Right? In my first run, Sp2 amplified on cycle 31.7. If I add 10-times more cDNA to the next reaction, Sp2 should amplify on cycle 28.4, Sp4 on cycle 35.0 and Sp5 as late as cycle 41.6, which is obviously too late.
What should I do?