Biorad iq cycler

DNA-free total RNA was extracted from left myocardial samples by a standard protocol with the RNeasy kit and RNase-free DNAse Set (Qiagen, Hilden, Germany). cDNA synthesis was carried out with iScript cDNA synthesis kit (BioRad, München, Germany) according to manufacturer’s instructions. QRT-PCR was performed on a Biorad iQ-Cycler using SYBR Green Supermix (BioRad). Primer sequences were as follows: Hprt: 5′-TCCTCCTCAGACCGCTTTT-3′(sense) and 5′-CATAACCTGGTTCATCATCGC-3′ (antisense); Cox I: 5′-TCGAAGGAGTCTCTCGCTCT-3′(sense) and 5′-CTGGTTCTGGCACGGATAGT-3′(antisense); CoxII: 5′-CAAGACAGATCATAAGCGAGGA-3′ (sense) and 5′-GGCGCAGTTTATGTTGTCTGT-3′ (antisense); Atp5a1: 5′-AAGCTGCAAGGATGCTGTCT-3′ (sense) and 5′-CAACAAAGGATGACCCCAAA-3′ (antisense); Uqcrc1: 5′-AGACCCAGGTCAGCATCTTG-3′ (sense) and 5′-CAGCGTCAATCCACACTCC-3′ (antisense).

The expression of specific genes in brain tissue was measured on days 7 and 14 post-TBI. Several brain areas of the ipsilateral hemisphere were sampled, including the cortex at the injury site, hippocampus, and striatum. The samples were placed in RNAlater (Thermo Fisher Scientific) for RNA stabilization. Total mRNA was isolated using the RNeasy Mini Kit (Qiagen, Venlo, Netherlands) and cDNA synthesis was carried out with the RevertAid™ First Strand cDNA Synthesis Kit (Thermo Fisher Scientific). The real-time polymerase chain reactions were set with qPCRmix-HS SYBR mastermixes (Evrogen, Moscow, Russia) and conducted in Bio-Rad iQ cycler (Bio-Rad). The program used initial denaturation at 95 °C for 5 min followed by 40 cycles of denaturation (95 °C, 20 s), primer annealing (56–63 °C, 20 s), and elongation (72 °C, 20 s). The data were normalized to averaged values for two reference genes, GAPDH and ACTB, using the ΔC (T) method. The gene-specific primers (Supplemental Table S2) were designed in NCBI Primer-Blast.

Real-Time PCR was performed in a Bio-Rad iQ-cycler (Bio-Rad) equipped with skirted Micro seal 96-Well PCR-plates covered with Microseal ‘B’ adhesive foils (Bio-Rad). Reaction volume (20 μl, containing 30 ng cDNA) consists of 4 μl diluted cDNA-solution (7.5 ng/μl), 2 μl 0.1 μM gene-specific forward and reverse primer solution (2 pMol each; Biomers, Ulm, Germany), 4 μl DEPC-treated water and 10 μl RT^{2 (link)} Real-Time SYBR-Green/Fluorescein-PCR-Master-Mix (SA Bioscience, Hilden, Germany/Qiagen). According to manufacturer recommendations, a cycling program with an initial activation step (94 °C, 3 min) followed by 45 cycles of denaturation (94 °C, 30 s), annealing (60 °C, 30 s), elongation (72 °C, 30 s) and a final elongation (72 °C, 30 s) is executed. Fluorescence acquisitions in the SYBR green and ROX (internal reference dye) channels were performed at the end of the annealing step. A melting protocol ranged from 94 to 59 °C following a stepwise increment of 0.5 °C held for 3 s. Each sample as well as a negative template control (NTC) was amplified in triplet for each of the primer pairs assayed. Raw data (ct-values) were extracted using iCycler iQ-software (version 3.1, Bio-Rad) running on the Bio-Rad iQ-cycler.