Random hexamer primers

HepG2, Caco-2 and MCF-7 cells were exposed to AhR agonists or coffee in either PBS or AHS for 4 h, in 12-well plates. Three wells of cells received each treatment. Total RNA was extracted using TRIzol (Invitrogen, Carlsbad, CA, USA) and was treated with RNase-free DNase (Promega). Reverse transcription was performed using the extracted total RNA, random hexamer primers (Amersham, Uppsala, Sweden), dNTPs, RNAsin (Promega), and Moloney murine leukemia virus reverse transcriptase (Promega). The cDNA thus obtained was subjected to quantitative real-time PCR, which was performed using a LightCycler TaqMan Master kit (Roche Applied Science, Penzberg, Germany) and a LightCycler 480 (Roche Applied Science). The PCR conditions were set according to the manufacturer’s instructions. The primers for human CYP1A1 (5′-TCCAAGAGTCCACCCTTCC-3′ and 5′-AAGCATGATCAGTGTAGGGATCT-3′) and the probe used for detection (#83; Roche Applied Science) were determined using the Roche Applied Science Universal ProbeLibrary Assay Design Center (http://www.roche-applied-science.com). For each sample, real-time PCR amplification of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was performed simultaneously using the Universal ProbeLibrary Human GAPD Gene Assay (Roche Applied Science), and the CYP1A1/GAPDH ratio was calculated.

For sequence analysis, viral RNA was prepared from plasma by using a RNA extraction kit (Qiagen, Valencia, CA, USA), followed by reverse transcription with SuperScript III reverse transcriptase (Invitrogen) and random hexamer primers (Amersham Pharmacia, Piscataway, NJ, USA). For the single genome amplification (SGAs), cDNA was titrated through nested-PCR of HIV-1 env of 9 replicates/dilution. The dilution that provided 2/9 (less than 30%) positive reactions was used to generate HIV-1 env single genome amplicons [34 (link),35 (link)]. Sanger sequencing of HIV-1 env single genome amplicons, as previously described [36 (link)], was performed by the Central Analytical Facility at Stellenbosch University. Contigs of Sanger reads were assembled with CLC Sequence Viewer 6.7.1 software (CLC bio A/S, Aarhus, Denmark). At least 10 independent contigs per time-point were assembled.

Total RNA was prepared using RNeasy mini kit (Qiagen) and treated with DNase I, Amp Grade (Invitrogen, Carlsbad, CA, USA) prior to cDNA synthesis. RNA integrity was electrophoretically verified by ethidium bromide staining. RNA (1 μg) was reverse transcribed with 100 U of Superscript II plus RNase H⁻ Reverse Transcriptase (Invitrogen) using 100 μM random hexamer primers (Amersham Biosciences, Piscataway, NJ, USA), according to the manufacturer's instructions. Real-time quantitative PCR was carried out on a Bio-Rad CFX-96 detection system with quantitative qPCR SYBR Green reagents (Bio-Rad, Hercules, CA, USA) and with a primer concentration of 0.5 μM. For a list of primer sequences, see Supplementary Table 1. PCR conditions were standardized to 39 cycles of: 95°C for 08 s, 59°C for 05 s and 72°C for 10 s.