Rt2 sybr green rox qpcr master mix kit

Total RNA was extracted by (Sigma; #RTN350-1KT). cDNA was generated using QuantiTect Rev. Transcription Kit (#205313; Qiagen). qPCR was performed using RT² SYBR Green ROX qPCR Mastermix Kit (#330523; Qiagen). qPCR assay was performed on ABI Quantstudio 5 machine and normalized to Gapdh.
Primers used for qPCR are listed as follows: Gapdh: 5′-F-TCA​CCA​CCA​TGG​AGA​AGG​C-3′, 5′-R-GCTAAGCAGTTGGTGGTGCA-3′; Fn1: 5′-F-CCC​AGC​TCA​CTG​ACC​TAA​GC-3′, 5′-R-TTCTCCTGCCGCAACTACTG-3′; Inhba: 5′-F-GGA​GAT​AGA​GGA​CGA​CAT​TGG​C-3′, 5′-R-CTGGTTCTGTTAGCCTTGGGG-3′; Itga5: 5′-F-CTT​CTC​CGT​GGA​GTT​TTA​CCG-3′, 5′-R-GCTGTCAAATTGAATGGTGGTG-3′; Acvr1b: 5′-F-GTG​GGG​ACC​AAA​CGA​TAC​ATG-3′, 5′-R-CTGGTCACATACAACCTTTCGC-3′; Acvr2a: 5′-F-GGG​ACG​CAT​TTC​TGA​GGA​TAG-3′, 5′-R-GCCATTCCTGCATGTTTCTGC-3′;

Total RNA was extracted using an RNA-isolation Kit (Qiagen, 74106) and cDNA was produced using reverse transcription supermix (Bio-Rad, 1708841). Real-time quantitative polymerase chain reaction (RT-qPCR) for all targets was performed on a QuantStudio 6 Flex Real-Time PCR Systems using RT2 SYBR®Green Rox qPCR master mix Kit (Qiagen, 330521). The relative quantification of the target gene was analyzed using the 2^−ΔΔCt method. GAPDH was used as an internal control.

The cDNA was prepared using an iScript gDNA Clear cDNA Synthesis Kit (BioRad). Quantitative RT-PCR was performed using RT2 SYBR Green ROX qPCR Master Mix kit (Cat#330522, Qiagen) for Oct4 exon 1 primers or SensiFAST™ SYBR NO-ROX Mix (Bioline) for 18s RNA and lncRNAs. The qRT-PCR primers for Oct4 exon1 and 18s RNA were previously described (Cherepanova et al., 2016 (link)). For lncRNAs, we used primers as ENSMUST00000140952 (F: CACCCCAGCCCGGTAAAC R: CAT​GGG​CGA​TCC​ACA​TGA​A) and ENSMUST00000173605 (F: TTC​CTT​CTG​CGT​CAG​TAT​CAT​CTT R: CCA​AGC​TGC​TTT​GCT​CAA​ATT).