Miscript sybr green pcr assay

Qiagen miScript SYBR Green PCR assay was used for miRNA quantification in accordance with the manufacturer's instructions. In brief, the reverse transcription of miRNAs from the plasma samples, cell culture supernatants and tissues were performed with miScript II RT kit. The specific primers of miRNAs were designed by Qiagen, and exogenous cel-miR-39-3p was used as control. qRT-PCR was performed in triplicate using the CFX Connect™ Real-Time System (Bio-Rad, USA) under the following conditions: 95°C for 15 min, 40 amplification cycles of 94°C for 15 s, 55°C for 30 s, and 70°C for 30 s. The data are presented as 2^−ΔΔCT. In the analysis of exosomal miRNAs, the first sample collected in the healthy controls was used as 1 for normalization. In the analysis of miRNAs of the tumor tissues, the first sample collected in the low-risk patients was used as 1 for normalization.

Total rna extracted from plasma and cells using the miRNeasy kit (Qiagen) was based on the protocol of the manufacturer. C. elegans miR-39 (cel-miR-39-3p) mimics were loaded in plasma as Spike-in control. For mature miRNA quantification, cDNA was generated with reverse transcription using miScript II RT kit (Qiagen) and Cdna, which was further utilized as a template for real-time PCR. Expressions of human miR-374a-5p, miR-19a-3p, miR-106b-5p, miR-26b-5p and miR-20a-5p were then quantified by miScript SYBR Green PCR assay (Qiagen), and finally were normalize by cel-miR-39-3p in plasma and RNU6 in cells (Qiagen), respectively. Triplicate assays were performed for each sample on Step One-Plus machine (ABI).