I was recently performing a great deal of qPCRs on adipose tissue derived RNA. I had experimental samples from sick, and control samples from healthy individuals. I used the same chemistry for all samples. Yet for healthy controls I got huge variations in reference genes Cq's (Actin and GAPDH, both) for about one third of the samples (up to 6 cycles difference, whereas for experimental samples I got the variation within 1 cycle). The concentrations of RNAs are not very high, but allowed me to use 50 ng per each RT. The qualities are fine (high RINs), the purities are acceptable (260/280 ~2). What could go wrong? I have multiple samples per patient so I only tested two people, but it was similar for both. I am using Biorad CFX 384 with ThermoFisher Fast qPCR MasterMix and TF TaqMan probes (validated). What could be the cause? Have anyone encountered such an issue?
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