Sybr green mix

Quantitative reverse transcription polymerase chain reaction (qRT-PCR) was performed to assess the reproducibility and reliability of the differentially expressed miRNAs identified by small RNA sequencing analysis. Total RNA (1 μg) was reverse transcribed (RT) to cDNA using Bulge-Loop miRNA qRT-PCR Starter Kit (RiboBio Co., Ltd., China) in RT reaction (42 °C, 1 hour, 70 °C, 10 minutes, 4 °C∞), according to the manufacturer's instructions. The cDNA was amplified using miRNAs assay primers and the SYBR Green Mix (RiboBio Co., Ltd., China) according to the manufacturer's instructions on the Roche Lightcycler 480. The primer sequences are presented in Table 1. In addition, a common reverse primer was used in this experiment as follow: 5′-CAGTGCGTGTCGTGGAGT-3′. Each reaction was performed under the following conditions: initialization for 10 minutes at 95 °C, and then 40 cycles of amplification, with 2 seconds at 95 °C for denaturation, 20 seconds at 60 °C for annealing, and 10 seconds at 70 °C for elongation. All cDNA samples were quantified in triplicate and mean cycle threshold (Ct) was calculated from the duplicate PCRs. In addition, we included a no-template control and no-reverse transcriptase control in each run. The relative miRNAs expression levels were normalized to U6 snRNA expression and the fold change between 2 groups was calculated using the 2^-ΔΔCt method.

miRNA expression was detected using a Bulge-Loop miRNA qRT-PCR Starter Kit (RiboBio Co., Ltd, Guangzhou, China), according to the manufacturer’s instructions. Briefly, 1 μg miRNA was used for RT in a polymerase chain reaction (PCR) thermocycler at 42°C for 42 min and 70°C for 10 min. A quantitative real-time PCR (qPCR) reaction mix (20 ul) containing 2 ul RT product, 10 ul SYBR Green mix, 0.8 ul primers and 6.4 ul distilled water (RiboBio Co., Ltd, Guangzhou, China) was prepared, and amplification was carried out on a Roche LightCycler 480 instrument (Roche, Mannheim, Germany). cel-miR-39 was used as a spike-in reference, as is common practice (23 (link),24 (link)). The delta cycle threshold (ΔCt) of miRNAs of interest was calculated using the following equation: ΔCt = Ct[miRNA of interest] - Ct[cel-miR-39]. miRNA expression was calculated using the 2^-ΔΔCt method with an arbitrary value of 1 for the control group.

RNA of platelets was extracted using Trizol reagent (Invitrogen), quantified using a NanoDrop spectrophotometer and treated with RNAase-free DNase I (Invitrogen) as recommended by the manufacturer. For miRNA expression analysis, 0.5 μg RNA was added a polyA tail using miDETECT A Track™ miRNA qRT-PCR Starter kit (RiboBio, Guangzhou, China), and reverse transcribed with miDETECT A Track™ Uni-RT primer. Specific primers and SYBR green mix used to detect miRNAs were purchased from RiboBio Co., Ltd. The U6 endogenous control was used for normalization. For mRNA expression analysis, 0.5 μg RNA was reverse transcribed using a Superscript RT reagent kit (Takara). Specific primers for real-time PCR were listed in Table 2. β-actin was used for internal standard of mRNA quantification. The cycling conditions of mRNA qRT-PCR were as follows: 95°C for 30 s, followed by 40 cycles of 95°C for 15 s and 60°C for 30 s. Melting curve analysis was used for primers specificity detection.