Sybr green

RNA from breast cancer cells was extracted according to the manufacturer's instructions (Qiagen, Valencia, CA) and cDNA was generated with qScript cDNA synthesis kit (Quanta BioSciences, Gaithersburg, MD), followed by qPCR using SYBR green (Quanta BioSciences, Gaithersburg, MD) on an iCycler5 (Bio-Rad, Hercules, CA). Amplification of 36B4, a housekeeping gene, was used for normalizing gene expression values.

Quantitative transcriptional assays for the indicated inc genes were performed as described previously (Ouellette et al., 2014b (link)). Briefly, total RNA was collected from C. trachomatis L2 infected HeLa cells at the indicated times using Trizol (Invitrogen) and treated with Turbo DNAfree (Ambion, Life Technologies) to remove contaminating DNA, according to the manufacturer's guidelines. One μg DNA-free RNA was reverse-transcribed with random nonamers (New England Biolabs, Ipswich, MA) using SuperScript III RT (Invitrogen) according to the manufacturer's instructions. Equal volumes of cDNA were used in qPCR reactions with SYBR Green (Quanta Biosciences, Gaithersburg, MD) and measured on an ABI 7300 system (Applied Biosystems, Life Technologies). Duplicate DNA samples were collected from the same experiment using Dneasy Tissue kit (Qiagen). Chlamydial genomes were quantified from equal amounts of total DNA by qPCR as above and used to normalize transcript data as described (Ouellette et al., 2005 (link), 2006 (link)).

Total RNA from lung was extracted using a modified TRIzol (Invitrogen, Carlsbad, CA) protocol and spectrophometrically quantitated. The integrity of RNA was verified by formaldehyde agarose gel electrophoresis. Equal amounts (1 µg) of RNA from each sample were reverse-transcribed into cDNA with the oligo (dT) SuperScript II First-Strand Synthesis System for RT-PCR (Invitrogen). qRT-PCR was performed using 100 ng sample RNA and SYBR Green (Quanta Biosciences, Gaithersburg, MD) in a Bio-Rad CFX96™ Touch Real-Time PCR Detection System. Results were calculated and graphed using the ΔCT of the target gene and normalizer, β-actin. The primers’ sequences were as follows: IFN-γ forward 5′- GGTCATTCAGATGTAGCGG-3′; IFN-γ reverse 5′- CACTCTCCTCTTTCCAATTC-3′; β-actin forward 5′- GCCAACCGCGAGAAGATGACC-3′; β-actin reverse 5′- CTCCTTAATGTCACGCACGATTTC-3′; CXCL9 forward 5′-TGTGGAGTTCGAGGAACC CT-3′; CXCL9 reverse, 5′-TGCCTTGGCTGGTGCTG-3′; Granzyme B forward 5′-TGTTTTCTCTGCCATCTGCTCTC; Granzyme B reverse 5′- GCTTTGTAAAAGTCTCCAGCCTGTG-3′; CXCL10 forward 5′-GGTCCGCTGCAACTGCATCC-3′; CXCL10 reverse 5′- GCAATTAGGACTAGCCATCC-3′; IAV M1 Protein forward 5′- ATGAGCCTTCTAACCGAGGTC-3′; IAV M1 Protein reverse 5′- TGGACAAAACGTCTACGCTGCAG-3′.

Total RNA was isolated from cells by using a Qiagen RNeasy kit and then reverse transcribed to generate cDNA with the High Capacity cDNA kit (Applied Biosystems). Quantitative PCR was performed by using SYBR green (Quanta Biosciences) in an Applied Biosystems Step One Plus instrument. Results were normalized to SETDB1, a control mRNA found to not change expression from resting B cells through EBV-immortalization (Price et al., 2012 (link)) or GNB2LI (Bazot et al., 2015 (link)).

RNA was extracted using Trizol Reagent (Invitrogen). cDNA was synthesised using iScript cDNA Synthesis Kit (Bio-Rad). RT–PCR was performed using Quanta Biosciences SYBR Green. Primers: PRKDC_Fwd GAGAAGGCGGCTTACCTGAG, PRKDC_Rvr CGAAGGCCCGCTTTAAGAGA, IGF2R_Fwd AGCGAGAGCCAAGTGAACTC, IGF2R_Rvr TCGCTGTAAGCAGCTGTGAA, CAD_Fwd AGGTTTGCCAGCTGAGGAG, CAD_Rvr TAATGAGTGCAGCAGGGGTG, ATR_Fwd GGAGGAGTTTTGGCCTCCAC, ATR_Rvr TGTGGCACTGCCCAGCTC, GCN1_Fwd CTTGTGCCCAAGCTGACAAC, GCN1_Rvr GCCCTGTGTCATCCTCTACG, MTOR_Fwd GAAGCCGCGCGAACCT, MTOR_Rvr CTGGTTTCCTCATTCCGGCT, CMTR1_Fwd CATTGCCCCATTTCACATTTGC, CMTR1_Rvr TCTTAGGCCCTGTGCATCTG.