Luna universal qpcr reagent

Total RNA of fpEC was isolated using RNeasy Mini kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. Cell monolayer was washed using PBS, and 0.7 mL of QIAzol lysis reagent (Qiagen, Germantown, MD, USA) was added to lyse the cells, and RNA was isolated using the kit. The concentration and the purity of obtained RNA was quantified by measuring 260/280 ratio using the Scandrop 250 (Analytik Jena Jena, Germany), and RNA integrity was examined by gel electrophoresis. cDNA was made using Luna universal reverse transcriptase PCR kit (New England Biolabs Frankfurt, Germany). Real-time quantitative PCR (qPCR) was carried out by using Luna universal qPCR reagent (New England Biolabs, Frankfurt, Germany) in CFX384 or CFX96 real time PCR cycler (Bio-Rad Laboratories, Vienna, Austria). All primer sequences used in this study are listed in Table 1. All primers were designed by crossing the exons to avoid the amplification of genomic DNA. Primer efficiency was verified by standard calibration curves. Gene expression levels were normalized to HPRT1 (Hypoxanthine Phosphoribosyl transferase 1) house-keeping genes, and the results were calculated using the 2^−ΔΔCT method.

Total RNA was collected from cultured cells treated with 100 nM 5-aza-2′-deoxycytidine (decitabine; Sigma) or splenocytes from a naïve C57BL/6 mouse using NucleoSpin RNA Plus (Takara). RNA was reverse transcribed with SuperScript III RT and random primers (Invitrogen) to synthesize first-strand cDNA. RT-qPCR was performed with Luna Universal qPCR reagent (NEB). eMLV env was detected with 5’-AGGCTGTTCCAGAGATTGTG-3’ and 5’-TTCTGGACCACCACATGAC-3’ and 18S rRNA was detected with 5’-GTAACCCGTTGAACCCCATT-3’ and 5’-CCATCCAATCGGTAGTAGCG-3’ on Roche LightCycler 480 II (104 (link), 105 (link)).

Whole genome DNA was extracted from exponential cell culture by GenElute Bacterial Genomic DNA Kit (Merck). Approximately 10 ng of DNA was subjected to quantitative PCR using the CFX384 Real-Time System (Bio-Rad, Hercules, CA, USA) with the SYBR Green I Master reagent (Roche, Basel, Switzerland) or Luna Universal qPCR reagent (New England Biolabs, Ipswich, MA, USA). narW locus was used for chromosome quantification and repZ, tetR and orf7_R388 were used for pESBL, F and R388 plasmid quantification, respectively. Plasmid DNA containing both the chromosomal and plasmid fragments (pEYY3, pEYY161 and pEYY413 for pESBL, F and R388, respectively) was used to draw standard curves.