Serum plasma mirna isolation kit

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.

Serum samples from athletes (pre- and post-season) along with healthy controls were used to isolate total RNA. RNA was isolated using 100 μL of serum using a serum/plasma miRNA isolation kit (Qiagen, Hilden, Germany) as per the manufacturer's recommended protocol. RNA was eluted in 20 μL of DNAse RNAse free water and stored at −80°C for further use. For a quality check of RNA, a bioanalyzer assay using a small RNA assay was performed to confirm the quality of RNA.

Serum samples from athletes were used to isolate total RNA. RNA was isolated using 100 lL of serum using a serum/plasma miRNA isolation kit (Qiagen Inc.) as per manufacturer’s recommended protocol. The RNA was eluted in 20 lL of DNAse–RNAse-free water and stored at −80C for further use. For a quality check of the RNA, a bioanalyzer assay using a small RNA assay was performed to confirm the quality of the RNA.