Hs rnu6b 2 miscript primer assay

To find out the potential microRNA binding sites, 3'UTR region of a gene was scanned using TargetScan (www.targetscan.org/), miRBase (www.mirbase.org/), microRNA.org (www.microrna.org/), RegRNA (www.regrna.mbc.nctu.edu.tw/) and MicroCosm (www.ebi.ac.uk/enright-srv/microcosm/). Target microRNAs were selected based on its conserved seed match or seed match with a higher context score. Total RNA (1μg) isolated from tissue samples were reverse transcribed using miScript PCR starter kit (Qiagen) according to manufacturer’s protocol and microRNAs were quantified using miScript SYBR Green PCR kit and 10X miScript Primer Assay (specific for microRNA of interest, Qiagen). U6 small nuclear 2 was quantified to normalize the levels of microRNA expression using Hs_RNU6B_2 miScript Primer Assay (Qiagen).

qRT-PCR for miR-185 targets and hypertrophic markers was performed with primers listed in Table B in S1 File, using SYBR green dye (Kapa Biosystem) and StepOne Plus Real Time PCR System (Applied Biosystems). miRNA-specific qRT-PCR in tissue or isolated cells was done using miScript SYBR Green PCR Kit (Qiagen) according to the manufacturer’s protocol, with miScript Primer Assay (for miR-185; Qiagen). The expression of the mRNAs was normalized to 18S rRNA and the level of miR-185 was normalized to U6 small RNA using the Hs_RNU6B_2 miScript Primer Assay (Qiagen). All reactions were performed in triplicate.

Analysis and quantification of mRNA expression levels were performed with the primers listed in Supplementary Table S3, using SYBR green dye (Kapa Biosystems, Boston, MA, USA) and StepOne Plus Real Time PCR System (Applied Biosystems, Waltham, MA, USA). miRNA-specific qRT-PCR in tissue or isolated cells was performed using a miScript SYBR Green PCR kit (Qiagen) according to the manufacturer’s instructions, with miScript Primer Assay (Qiagen). The expression of mRNAs was normalized to 18S rRNA and the level of mature miRNA was normalized to U6 small RNA using the Hs_RNU6B_2 miScript Primer Assay (Qiagen), respectively. All reactions were performed in triplicate.