Snord61 miscript primer assay

Mature miRs were analyzed by miScript II RT kit (Qiagen) and miScript SYBR Green PCR kit with miScript Primer Assay kit (Qiagen) according to manufacturer’s instructions. Briefly, RNA was isolated from left ventricles and cDNA for mature miR profiling was prepared using the miScript II RT kit. Mature miRa were determined by real-time PCR using the miScript SYBR Green PCR kit (Qiagen). cDNA template was diluted to 1 ng/μl in RNase free water. Two nanograms of template cDNA were used for miRs quantification in a final volume of 25 μl system containing specific primers and QuantiTect SYBR Green PCR master mix following manufacturer’s instructions. Primers included miScript Universal Primer, rat-specific miScript mature miRNA primers and SNORD61 miScript Primer Assay (Qiagen). Serial dilutions of the positive control were done on each plate to create a standard curve for the quantification. The real-time PCR was performed in triplicate and threshold cycle numbers were averaged for each sample using IQ5 real-time PCR (BioRad). The expression levels of each mature miR in control and losartan-treated heart tissues were computed following the method described by Livak and Schmittgen,[27 (link)] and expressed as fold of SNORD61.

The levels of miR-210 were detected as we previously described [32 (link), 33 ]. Total RNA was extracted using the TRIzol reagent (15596026; Invitrogen). MiR-210 levels were analyzed by miScript II RT kit (218161, Qiagen) and miScript SYBR Green PCR kit (218073, Qiagen) with miScript Primer Assay kit (MS00000644, Qiagen) according to the manufacturer’s instructions. Briefly, 1 μg of template RNA was mixed with reverse-transcription master mix in a final volume of 20 μl and incubated at 37 °C for 60 min, and then the reaction was stopped at 95 °C. Two nanograms of template cDNA was used for miR-210 quantification in a final volume of 25 μl system containing specific primers and QuantiTect SYBR Green PCR master mix according to the manufacturer’s instructions. Primers included miScript Universal Primer, miR-210 miScript Primer Assay, and SNORD61 miScript Primer Assay (MS00033705, Qiagen). PCR was done in triplicate and threshold cycle numbers were averaged for each sample. The relative expression levels of mature miR-210 were calculated using the formula 2(^-ΔΔCt) and normalized to SNORD61. The change of miR-210 was expressed as fold of normal control.