Abi 7500 fast real time pcr cycler

Total RNA was isolated with peqGOLD Total RNA Kit (PeqLab) and reverse transcribed using the Superscript II RT First Strand Kit (Invitrogen). Real time PCR was conducted in the ABI 7500 Fast Real-Time PCR cycler (Applied Biosystems) using a SYBR Green I Low Rox Mastermix (Eurogentec GmbH) and the respective primers (Supplementary Table S2). Following a 10 min denaturing step at 95°C 40 cycles with 15 seconds 95°C and 1 min 60°C were applied. Primer sequences and PCR efficiencies are given in Supplementary Table S2.

Differentiated myotube RNA was extracted and purified following the Direct‐zol RNA Miniprep Kit protocol (ZYMO Research, Orange, CA). The concentrations of RNA were determined using NanoDrop spectrophotometer (Thermo Fisher Scientific, Wilmington, DE). RNA (200 ng × μL⁻¹) was reverse transcribed into cDNA according to the manufacture's protocol of High Capacity cDNA Reverse Kit (Applied Biosystems, Foster City, CA). Real‐time qPCR was carried out with the ABI 7500 Fast Real‐time PCR cycler (Applied Biosystems). Primer sequences are presented in Table 1. An endogenous control gene (18 S) was used as an active reference to normalize quantification of an mRNA target. Relative mRNA expression levels were determined from the cycle threshold (Ct) values using the 2^−ΔΔCt comparative method.

Relative expression levels of the HSP70 isoforms were determined by SybrGreen real time PCR applying the comparative ΔΔ−C_T method. Total cellular RNA was isolated using the RNAeasy kit (Qiagen, Hilden, Germany) with a subsequent DNaseI digestion step according to the manufacturer's instructions followed by cDNA synthesis with the Superscript II RT First Strand Kit (Invitrogen GmbH, Karlsruhe).
Real time PCR was conducted in the ABI 7500 Fast Real-Time PCR cycler (Applied Biosystems Inc., Foster City, CA, USA). The standard PCR reactions (20 μl) contained 1 μl cDNA and 10 μl 2× SybrGreen I Low Rox Mastermix (Eurogentec GmbH, Cologne, Germany) for detection. The thermal cycling conditions comprised an initial denaturation step at 95 °C for 10 min, followed by 40 cycles of three-step PCR including 15 sec at 95 °C, 60 sec at 60 °C and 30 sec at 95 °C. The C_T levels of the investigated HSP70 isoforms were normalized to GAPDH (Δ-C_T level). Δ-C_T was calculated as C_{T HSP isoform, sample} - C_{T GAPDH, sample}.
Primer pairs for the different HSP70 isoforms were selected from NCI qPrimerDepot (Table 1) (http://primerdepot.nci.nih.gov/).