Mirna serum plasma kit

RNA was isolated from P21 pellets of 0.5, 1.0, 1.5, 3.0, 4.5, 9.0, and 13.5 mL urine fractions that were resuspended in 0.1 mL of 1X PBS using miRNA serum/plasma kit (Qiagen). Cel-miR-39 was spiked in the qiazol solution as per manufacturer’s instructions. For the SN21^TCEP as well, the RNA was isolated using the same kit. RNA concentration was measured using Nanodrop 2000 (Thermo Fisher Scientific). Reverse transcription was performed using Taqman miRNA reverse transcriptase kit (Thermo Fisher Scientific). Primers for miR-16, miR-155, miR-200b, miR-203, and Cel-miR-39 were used for reverse transcription followed by real time QPCR (Quantstudio3, Thermo Fisher Scientific). The results were then exported and the Ct values for each of the miRNA were normalized against the spike-in control (Cel-miR-39) and the obtained dCt values for the urine P21 pellets from various fractions were compared.

For publicly available data previously published by us, raw sequencing reads were downloaded from NCBI GEO, Study GSE151362 [24 (link)] and combined with new small-RNA dataset with sequencing as follows. miRNA was isolated from 200 μL plasma samples using the Qiagen miRNA serum/plasma kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions and stored at −80 °C.
Library preparation and sequencing was performed using Qiagen (Valencia, CA) with the QIAseq miRNA Library and QIAseq miRNA 48 Index IL kits as per the manufacturer’s instructions (Qiagen, Hilden, Germany). Amplified cDNA libraries underwent single-end sequencing by synthesis (Illumina v1.9) using the Illumina NovaSeq 75 bp single-end read sequencing on miRNA libraries from 42 plasma samples taken from 10 non-pregnant and 32 pregnant women aged 16 to 44 years.