Jumpstart taq dna polymerase enzyme

Total RNA was isolated using the RNeasy Plus Mini Kit (Quiagen, USA), and cDNA was synthesized from 1 μg of RNA via a High Capacity RNA-to-cDNA Kit (Life Technologies, USA) following the manufacturer’s instructions. Gene expression was analyzed by quantitative real-time PCR (qPCR) using MyiQ real time PCR System (Bio-Rad, USA). The applied primers (Supplementary Table 3) were designed by Oligo software (Oligo Perfect Designer, Invitrogen, USA). Reactions were measured in duplicates using custom 2× Syber Green Master Mix based on the hot-start Jumpstart Taq DNA Polymerase enzyme (Sigma, USA). The amplification was done for 40 cycles (95 °C 20 sec, 60 °C 20 sec, 72 °C 40 sec). To verify the PCR products, melting curves and negative controls were carried out in each reaction. Relative quantification of mRNA was determined by the ΔΔCt method (2^−ΔΔCt formula)19 (link) by using the expression profile of the corresponding control samples as reference.

Total RNA from the MuSCs, iMuSCs, and the neuronal differentiating iMuSCs were isolated using the RNeasy Plus Mini Kit (Qiagen, USA). Total RNA from the biopsied TA tissues was isolated using the RNeasy Microarray Tissue Mini Kit (Qiagen, USA). cDNA was synthesized from 1 µg of RNA via iScriptTM cDNA Synthesis Kit (Bio-Rad, USA) following manufacturer’s instructions. Gene expression was analyzed by quantitative real-time PCR (qPCR) using MyiQ real-time PCR (Bio-Rad, USA). The applied primers were designed by Oligo software (Suppl. Table 2) (Oligo Perfect Designer, Invitrogen, USA). Reactions were measured in duplicate using custom 2x Syber Green Master Mix based on the hot-start Jumpstart Taq DNA Polymerase enzyme (Sigma). The amplification was done for 40 cycles (95 °C 20 sec, 60 °C 20 sec, 72 °C 40 sec). To verify the PCR product, melting curves were carried out in each reaction. Relative quantification of mRNA was determined by the ΔΔCt method (2^−ΔΔCt formula)^{24 (link)} using the expression profile of the corresponding control samples as reference.