We are having trouble measuring viral genome copy numbers using qPCR. The CT values for dengue virus are quite varied; between technical replicates (same sample), we may see 10 CT differences and sometimes no detection. Has anyone had a similar experience and found a way to correct the issue?
We speculate it may be generating cDNA using Random Primers. We usually use oligo dT for cDNA synthesis but switched with viral infection to random primers. Previously, we have had no issues with qPCR and have gotten consistent CT values.
The Random primers we use are a stock concentration of 10uM, added in 1ul volume to a 20 ul cDNA reaction. This is the concentration recommended by the kit (https://www.abmgood.com/onescript-plus-cdna-synthesis-kit.html).
Any tips would be greatly appreciated!
You have tu use dT for the sinthesus o cDNA in special if you target is a expressed gene. Also you have to standarize your protocol as normal PCR then you have to do it your qpcr in 2 steps.
agree with Jack Greenhouse here. You can also do it as two step and use the reverse PCR primer for the cDNA synthesis.
Best Sven
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