Maxima h minus dna synthesis kit

Total RNA was isolated using the PureLink RNA Mini Kit (Thermo Fisher Scientific) and treated with DNase I (Thermo Fisher Scientific) according to the manufacturer’s instructions. Reverse transcription was carried out employing the Maxima H Minus DNA Synthesis Kit (ThermoFisher Scientific). The SYBR Select Master Mix (ThermoFisher Scientific) was used for qRT-PCR, following the manufacturer’s instructions. Reactions were conducted with the IQ5 rtPCR Detection System (BioRad, Munich, Germany). Transcripts of target genes were amplified using the gene-specific primers for human CFTR 5′-GGGCTGTGTCCTAAGCCATGGCCA-3′ and 5′-GATGGCTTGCCGGAAGAGGCTCC-3′. Absolute quantification was performed using a several fold dilution of target specific plasmid-DNA as internal standard curve. The resulting molecule concentrations were normalized to a reference gene (Mrps18a: mitochondrial ribosomal protein S18a). Constant expression of Mrps18a was confirmed against other common reference genes. The fold change of mRNA levels was calculated with the relative standard curve method. Measurements were performed in technical triplicates and six biological replicates. Melting curves and gel electrophoresis of the PCR products were routinely performed to determine the specifity of the PCR reaction.

Total RNA was isolated using the PureLink RNA Mini Kit (Thermo Fisher Scientific, Waltham, MA, USA) and treated with DNase I (Thermo Fisher Scientific) according to the manufacturer’s instructions. Reverse transcription was carried out employing the Maxima H Minus DNA Synthesis Kit (Thermo Fisher Scientific). Real-time quantitative PCR (RT-qPCR) was performed in the CFX 96 Real-Time system (Bio-Rad, Munich, Germany) using the SYBR Select Master Mix (Fisher Scientific GmbH) and gene-specific primers [41 (link),42 (link)] as described before [42 (link)]. The resulting molecule concentrations were normalized to a reference gene encoding for the mitochondrial ribosomal protein S18a (Mrps18a).