Validating miRNA Expression via Stem-Loop RT-qPCR

To validate the miRNAs identified by small RNA sequencing, a stem-loop reverse-transcription quantitative polymerase chain reaction (RT-PCR) assay was used to specifically detect mature miRNAs. The stem-loop RT-PCR primers were designed according to the previously reported methods [33 (link),34 (link)]. The samples for quantitative stem-loop RT-PCR were same as sequencing samples. 2 μg RNA of each sample was used for stem-loop RT-PCR. The reverse transcription was performed using specifically stem-loop RT-PCR primers and M-MLV Reverse Transcriptase (Promega, M1701). U6 was used as the endogenous control. The quantitative stem-loop RT-PCR analysis was performed using SYBR Green I PCR Master Mix (Roche, 4887352001), and the products were detected with the Light cycler 480 II (Roche). The relative quantification of miRNA expression was performed using the comparative CT (2^-△△CT) method. The data were presented as the mean ± SD of triplicated wells. The Student’s t-test was used to analyze the expression difference between the 2 groups. The primer sequences used are listed in S1 Table.