Reverse transcription (RT) based quantitative PCR (qPCR) for quantifying microRNAs
(miRNAs) in in the circulation presents specific challenges. Here, we describe an
optimized research protocol to assess serum sample quality and quantify levels of
a panel of four test miRNAs (miR-371a-3p, miR-372-3p, miR-3733p, and miR-367-3p) that
enables highly sensitive and specific malignant germ cell tumor (GCT) diagnosis and
monitoring. This protocol utilizes a multiplex RT step using Taqman miRNA stem-loop
primers. A multiplexed preamplification stage is then employed to increase the sensitivity
of the final quantification step, which is performed using standard singleplex Taqman
qPCR methodology.
