I have performed microRNA TLDA assay. In TLDA assay, I used U6 as internal control (moch and experiment U6 Ct is about 20). miR-XXX was confirmed using Single Taqman assays. I used U6 as internal control. In TLDA result, miR-XXX was overexpressed in 8-fold high compared with mock (raw Ct=33 vs. raw Ct=37 and delta-delta Ct is about 3). when I used Single Taqman assays to confirm this result, delta-delta Ct became 0.2.
When I perform RT reaction, I diluted total RNA to 10 ng total RNA/microliter and used 10 ng total RNA (1 microliter) to total 15 microliter RT reaction. Then I used 1.33 microliter cDNA to total 20 microliter real-time reaction.
In mock and experiment, miR-XXX Ct is about 22, and U6 Ct is about 21.
I recheck my diluent total RNA concentration is 13 ng total RNA/microliter (mock),and 10 ng total RNA/microliter (experiment). Although initial RNA input is different, U6 Ct is almost equal.
Can I ignore the difference of total RNA input in relative quantitative real-time PCR for microRNA if internal control Ct value is equal?
Thanks for your help and answer.
Total RNA input and internal control ct value
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