Gotaq 1pcr master mix

Total DNA and RNA were extracted from PBMCs using the QIAamp and RNeasy kits, respectively, according to the manufacturers’ instructions (QIAGEN, Germany), to assess the gene products by quantitative polymerase chain reaction (PCR). The purified RNA in mRNA samples was reverse transcribed into complementary DNA using the QuantiTect Reverse Transcription Kit (QIAGEN, Hilden, Germany). The PCR was performed in duplicate using the GoTaq1 PCR Master Mix (Promega, Madison, WI, USA) and the subsequent primers (Table 2) on a CFX Connect Real-Time System (Bio-Rad Labs, Hercules, CA, USA).
Relative mRNA expression was calculated after normalization using beta actin as internal controls utilizing the 2^−ΔCT method. The mitochondrial DNA copy number was quantified as the ratio of DNA products of mitochondrial nicotinamide adenine dinucleotide dehydrogenase subunit 1 normalized to ribosomal 18S-RNA serving as an internal control using the 2^−ΔCT method, as described previously [37 (link)].

RNA was isolated from 8-weeks-old mice testes using TRIzol (Invitrogen) according to the manufacturer’s instruction. 1 μg of total RNA and sorted cells was reverse transcribed using High Capacity cDNA Reverse Transcription Kit (Applied Biosystems) following the manufacturer’s recommendation. The resulting cDNA was used both for PCR using GoTaq Flexi DNA Polymerase (Promega) or quantitative PCR (qPCR) using GoTaq 1PCR Master Mix (Promega) with specific primers (Supplementary Table 1), according to the manufacturer’s instructions. For qPCR, relative mRNA levels were calculated in accordance with the point where each curve crossed the threshold cycle (Ct) from ABI Prism 7900HT (Applied Biosystems), subtracting the Ct of the 18S housekeeping gene (∆Ct) according to the formula: 2^{−(∆Ct problem−∆Ct control)}.

Immunoblot was performed as described [25 ]. For specific protein detection, membranes were incubated with anti‐TCFL5 (Sigma), anti‐SOX2 (Cell Signalling, Danvers, MA, USA), anti‐KLF4 (Cell Signalling), anti‐E‐cadherin (Cell Signaling) and anti‐HSP90 (Sigma) in 5% BSA‐TBST overnight at 4 °C. Complete membranes were shown in Fig. S6.
RNA was isolated and reverse transcribed as described [25 ]. cDNA was used both for PCR using GoTaq Flexi DNA Polymerase (Promega, Madison, WI, USA) or quantitative PCR (qPCR) using GoTaq 1PCR Master Mix (Promega) with specific primers (Table S1), according to the manufacturer’s instructions. For qPCR, values were normalized as described [25 ].