Mir 210 miscript primer assay rn mir 210 1

MiR-210 levels were determined by miScript II RT kit (Qiagen) and miScript SYBR Green PCR kit with miScript Primer Assay kit (Qiagen) according to manufacturer's instructions. Primers included miScript Universal Primer, miR-210 miScript Primer Assay (Rn_miR-210_1; Cat#MS00000644; Qiagen) and SNORD61 miScript Primer Assay (Hs_SNORD61_11; Cat#MS00033705; Qiagen). Briefly, 1 μg of template RNA was mixed with reverse-transcription master mix in a final volume of 20 μl and incubated for 60 minutes at 37°C, and the reaction was stopped at 95°C. Two nanograms of template cDNA were used for miR-210 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, miR-210 miScript Primer Assay 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. PCR was done in triplicate and threshold cycle numbers were averaged for each sample.