M mlv reverse transcriptase rnase h minus kit

Total RNA was extracted from cells grown in a monolayer in cell culture dishes with a kit following the instructions of the supplier (Promega, Heidelberg, Germany). First-strand cDNA synthesis was performed using M-MLV reverse transcriptase RNase H Minus-kit from Promega. Each sample for real-time RT-PCR analysis contained 200 ng of cDNA, SYBR Green Master Mix and 0.4 μM of each primer. The PCR amplification and real-time fluorescence detection were performed with the Opticon III instrument (MJ Research) using the QuantitectTM SYBR1 green PCR kit (Qiagen, Hilden, Germany). As quantification standard defined concentrations of annexinA7 cDNA were used for amplification. PCR amplification was carried out according to the manufacturer’s instruction and all PCR products were amplified in a linear cycle. GAPDH mRNA was employed as an internal standard, and expression of each gene was determined by RT-PCR and normalized against WT GAPDH mRNA levels. Data are the mean ± SD from three samples per group of four independent experiments. All primers are listed in Supplementary Table S1.

To quantify HERVFc-1 transcripts, 2 μg of total RNA and 100 μM Oligo-dT primer were used for cDNA synthesis (M-MLV Reverse Transcriptase, RNase H Minus-Kit; Promega GmbH, Walldorf, Germany) following the manufacturer’s instructions. A sample without reverse transcription was also prepared to check for genomic DNA contamination (noRT control).
For the qRT-PCR 5 μL 5xGreen GoTaq buffer, 0.2 μl GoTaq polymerase (both Promega GmbH, Mannheim, Germany), 16.8 μl nuclease-free water, 0.5 μl of 10 mM dNTPs (Thermo Fisher Scientific, Waltham, MA, USA), 0.25 μl forward primer (25 μM), 0.25 μl reverse primer (25 μM) and 2 μl cDNA were prepared. The amplification was performed under the following conditions: 94°C for 30 s, 60°C for 30 s, 72°C for 45 s (40 cycles). The experiment was repeated four times for the gene expression analysis.
Relative gene expression was calculated with the 2^-ΔΔCt method (51 (link)) using hypoxanthine phosphoribosyltransferase 1 (HPRT1) as reference gene for normalization. noRT controls were used for exclusion of genomic DNA amplifications. The following primers from Eurofins Genomics GmbH (Ebersberg/Germany) were used: HERVFc-1_forward: 5’-CTC CCC ATC TCT CTG GTG C-3’ and HERVFc-1_reverse: 5’-TGA GGA GGC TGG TTT CTC TAA G-3’; HPRT1 _forward: 5’-ACC AGT CAA CAG GGG ACA TAA-3’ and HPRT1_reverse: 5’-CTT CGT GGG GTC CTT TTC ACC-3’.