High CT Value for qRT-PCR to miRNA in cancer plasma

Question

[Shicheng Guo]

Hi All,

Any empirical experience on the CT value to miRNA detection in cancer plasma samples? Do you think the CT is as high as 40 is normal and acceptable? Suppose the miRNA concentration is very low, can I amplify miRNA with very low-cycle PCR and then detect them with another qRT-PCR??

Thanks.

[VHahaut]

Hi!

I never worked on plasma so my experience is mainly on cells/serum samples from such type of patients. However a Ct of 40 is usually considered as noise. Have you run any control such as water/RT negative samples that would allow you to set your limit of detection? Moreover, depending on your assay a melting curve could be great information about amplification of your product. Finally, I never tried to do a pre-amplification but an obvious biais of this would be for comparison to other miR since the amplification will lead to inbalance in the starting material (due to the way you amplify the miRs).