Rnase free dnase

Total RNA was isolated and purified using the RNeasy Mini Kit and RNase-Free DNase (Quiagen) as per manufacturer instructions. RNA concentration and quality were determined by spectrophotometry (Spark, Tecan, Männedorf, Switzerland). The RNA was directly used for gene expression analysis. A one-step quantitative polymerase chain reaction (qPCR) was performed, including the cDNA synthesis and subsequent qPCR detection. For this, 1 μg of RNA was mixed with the SensiFAST™ SYBR® Hi-ROX One-Step Kit’s Mastermix (Bioline, London, UK) and specific bovine primers listed in Table 1 (ThermoFisher Scientific, Waltham, MA, USA). The reaction mixture was denatured at 95°C for 10 min, followed by 40 cycles of 95°C for 15 s and 60°C for 1 min. Relative expression of target genes was calculated according to the 2^-ΔCt method (ΔCt = Ct_{(gene of interest)}−Ct_(GAPDH)), according to published guidelines [19 (link)].

RNA was extracted from the RNAlater preserved abomasal mucosa and gastric lymph node samples as described previously [45 (link)] using a Stratech Beadbeater-8 (Stratech Scientific, Soham, UK) and 1 mm³ zirconia-silica beads (Thistle Scientific, Glasgow, UK), RNeasy Mini Kit (Qiagen, Crawley, UK) with β-mercaptoethanol (Thermo Fisher Scientific Inc., Waltham, USA), Qiashredder columns (Qiagen, Crawley, UK) and Qiagen RNAse-free DNAse (Qiagen, Crawley, UK). Complete removal of native DNA contamination required additional DNAse treatment in solution (DNA-free kit, Applied Biosystems, Warrington, UK). The 260 nm absorbance of a 1:5 dilution of sample RNA was measured using a Cecil CE2021 2000 series spectrophotometer (Cecil Instruments, Cambridge, UK) and used to calculate the RNA concentration. RNA purity was assessed by determining the ratio 260/280 nm absorbance.

Total RNA was extracted using Ribospin^TM Plant kit (GeneAll Biotechnology Co., Ltd., Songpa-gu, South Korea) according to the manufacturer’s instructions. The RNA samples were treated with Qiagen RNase-Free DNase (Qiagen, 79254, Qiagen Inc., Midland, ON, Canada) for 30 min at 37°C to remove the genomic DNA. Following the manufacturer’s protocol, 1 μg of total RNA was used to make cDNA at a volume of 20 μl, using Thermo Scientific Revert-Aid^™ First-Strand cDNA Synthesis Kit (Fermentas, K1622, Thermo Fisher Scientific, Hudson, NH, USA).

Plasma was obtained following centrifugation of blood ethylenediaminetetraacetic acid-collection tubes. Peripheral blood mononuclear cells (PBMCs) were separated by gradient centrifugation in cell preparation tubes. CD4⁺ T cells were isolated from PBMCs using a CD4⁺ T-cell negative isolation kit and magnetic-activated cell sorting columns (Miltenyi Biotec, Teterow, Germany; purity >95%). One million CD4⁺ T cells were lysed in QIAgen lysis (RLT) buffer, and the lysates were stored at −80 °C until the RNA and DNA were extracted using the QIAgen AllPrep DNA/RNA Mini Kit (Cat.no. 80204, QIAgen, Melbourne, Australia). During RNA purification, RNA columns were treated twice with QIAgen RNase-Free DNase. Plasma HIV-1 RNA for sequencing was extracted from 2 to 3 ml of plasma by ultracentrifugation and a guanidinium-based method, including a prespin to sediment potential cell remnants [10 (link)]. HIV-1 RNA was extracted from 10 to 100 μl VOA supernatant using the same method as the plasma samples, but without the prespin. From cell-associated HIV-1 RNA, plasma HIV-1 RNA and VOA HIV-1 RNA, we generated complementary DNA using the Superscript III (Invitrogen by Thermo Fisher Scientific) cDNA synthesis kit and a gene-specific primer for p24-RT according to the manufacturer's instructions. See Supplemental Digital Content 2 for primers and PCR conditions.

Total RNA was isolated using the RNeasy Plus Mini Kit and treated with RNase-free DNase (both from Qiagen). Complementary DNA (cDNA) was synthesized using 1 μg of total RNA in 20 μL reaction mixture, containing random hexamers and Superscript III reverse transcriptase (Gibco, Invitrogen), according to the manufacturer’s instructions.
Taq polymerase together with Taqman probes (Thermo Fisher Scientific) for GAPDH (cat. no. 4333764F), NANOG (cat. no. Hs02387400_g1), POU5F1 (cat. no. Hs03005111_g1), MITF (cat no. Hs01117294_m1), BEST-1 (cat. no. Hs00188249_m1), RPE65 (cat. no. Hs01071462_m1), TYR (cat. no. Hs00165976_m1), PMEL (cat. no. Hs00173854_m1), MAP2 (cat no. Hs00258900_m1), PDGFRB (cat no. Hs01019589_m1), and TUBB3 (cat no. Hs00801390_s1) were used. Samples were subjected to real-time PCR amplification protocol on StepOne^TM real-time PCR System (Applied Biosystems). Three independent experiments were performed for every condition and technical duplicates were carried for each reaction. Results are presented as mean ± SEM (standard error of the mean).