Mirneasy serum plasma mirna isolation kit

The absolute expression (copy numbers) of 578 candidate miRNAs were quantified in each patient and control biospecimen using miRNA-specific RT-qPCR assays (MiRXES, Singapore) via a highly controlled workflow illustrated in online supplemental figure S1. The analytical specificity, reproducibility and sensitivity of the assay and workflow (online supplemental methods) are shown in online supplemental figure S2. Total RNA from 200 µL of patient and control serum specimen was isolated using miRNeasy serum/plasma miRNA isolation kit (Qiagen, Germany). Synthetic miRNA controls were added to samples before RNA isolation, and RT-qPCR to monitor and normalise technical variations throughout the entire workflow (online supplemental methods). Absolute expression of each miRNA was determined in each patient serum sample and normalised across samples using endogenous reference miRNAs (online supplemental methods).

Plasma miRNA was extracted using the miRNeasy serum/plasma miRNA isolation kit (Qiagen GmbH). The ID3EAL miRNA knowledge panel 384 Target kit (MiRXES) was used and experiments were performed according to the manufacturer's instructions. The kit contained the ID3EAL Panel RT Primer Pool for the miRNAs with a set of three proprietary spike-in RNAs [ID3EAL Panel RNA Spike-In (MiRXES)] to normalize variations in RNA isolation efficiency. Isolated serum RNA was subjected to RT using the ID3EAL Reverse Transcriptase kit (MiRXES). A 6-log serial dilution of synthetic templates for each miRNA was concurrently reversed-transcribed. Using the miRNA-specific qPCR assays-ID3EAL miRNA qPCR Master Mix (MiRXES), >300 candidate miRNAs were measured in each complementary DNA sample. The following cycling conditions were used: 95°C for 10 min, 40°C for 5 min, and then 45 cycles of 95°C for 10 sec and 60°C for 30 sec. Absolute copy numbers of each miRNA were determined by interpolation of the Cq values to that of the synthetic miRNA standard curves and adjusted for RT-qPCR efficiency variation. The 2^−ΔΔCq method was used for quantification (29 (link)).