2.3. miRNA validation by qRT-PCR

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 ​Table1.1. 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.

Primers used for qRT-PCR.