Miscript human serum plasma 384hc mirna pcr arrays

The Serum/Plasma miRNA isolation kit, miScript II RT Kit (using HiSpec buffer), and miScript SYBR Green PCR Kit (Qiagen) were used to prepare samples for profiling following standard protocols. The miScript Human Serum & Plasma 384HC miRNA PCR Arrays (Qiagen) were used to profile 372 miRNAs and 12 controls (6 snoRNA/snRNA controls, a miRNA reverse transcription control, a positive PCR control, and a C. elegans miR-39 spike-in) for each of the 48 serum samples. A Roche LightCycler 480 machine was used to perform qPCR. Using the miScript miRNA PCR Array Handbook, the handbook protocol determined the same baseline and threshold across all amplification plots, in which the earliest amplification was manually set to cycle 15 and the threshold was set to 0.1. To confirm qRT-PCR results from the first RNA isolation, RNA was isolated a second time from serum in separate tubes from the same participants. In this second experiment, miScript Primer Assays were ordered to separately confirm expression of six identified miRNAs of interest (please see Ct analysis and Results section), along with spike-in control (miR-39) and four stable miRNAs (miR-21, 122, 126, 574) by following standard protocols.