Primetime gene expression master mix

Total RNA was purified from the cells by using RNeasy Plus Mini Kit (Qiagen, Hilden, Germany) following the manufacturer’s instruction. cDNA was produced by reverse transcription of 1 μg DNA free RNA samples using SuperScript III First-Strand Synthesis System for RT-PCR Kit (Thermo Fisher Scientific, Waltham, MA, USA).
Quantitative real-time PCR assay was performed in 20 μL final volume containing 5 μL cDNA, 1× Prime Time Gene Expression Master Mix (Cat. No. 1055770, IDT, Coralville, IA, USA), 1× Prime Time qPCR assay (BCL2 (Cat. No. Hs.PT.56a.654557.g), GJA1 (Cat. No. Hs.PT.56a.38338544) and TUBA4A (Cat. No. Hs.PT.58.4392157.g); IDT)) using QuantStudio 12K Flex Software v1.2.2. Denaturation at 95 °C, 3 min was followed by 40 cycles (95 °C, 5 s and 60 °C, 30 s). Reactions were performed in triplicate using RNase-free water as negative control. CT-values were set in the exponential range of the amplification plots using the QuantStudio Detection Software. Relative expression levels were expressed as 2−ΔΔCT where ΔΔCT values correspond to the difference between the CT-values of the target and the TUBA4A internal control genes.

Qiagen DNeasy blood and tissue kit (Qiagen, Hilden, Germany) was used to extract genomic DNA from whole blood collected from previously ELISA-positive cows one week following milk ELISA screening. The SS1 qPCR assay, developed by CentralStar Cooperative Inc., is a multiplex probe-based quantitative PCR assay that targets the BLV proviral polymerase gene, bovine β-Actin gene, and an internal amplification spike-in control ultramer to quantify proviral load. Briefly, 3 µL of extracted DNA, 12.5 µL of 2 × PrimeTime Gene Expression Master Mix (ThermoFisher, Austin, TX, USA), 1.25 µL of a 20 × primer mix, 1 µL of internal spike-in control (10,000 copies/µL), and 7.25 µL of DNA-free water were combined for each qPCR reaction. All SS1 qPCR was performed on Applied Biosystems 7500 Fast Real-Time PCR system (FAST Real-Time PCR, Foster City, IA, USA) with qPCR conditions as follows: 95 °C for 10 min, 40 × (95 °C for 15 sec, 60 °C for 1 min). BLV and bovine β-Actin (a measure of host DNA) copy numbers were derived using a standard curve consisting of linearized plasmids containing respective target sequences previously quantified and normalized by digital droplet PCR. Amplification efficiency and manual thresholds were established from initial qPCR machine calibration. Proviral Load was calculated and expressed as the ratio between proviral BLV copies and bovine β-Actin copies.

RNA is extracted from cell pellets using the RNeasy Mini Kit (Qiagen, Hilden, Germany). Complementary cDNA is then synthesized using the High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Waltham, MA, USA). qPCR is performed using the Applied Biosystems StepOnePlus Real-Time PCR systems (Thermo Fischer Scientific). Primer sets were validated with transient temperature PCR and Platinum SYBR Green qPCR Supermix-UDG (Thermo Fischer Scientific). Following primer pairs were selected for LC3 (MAP1LC3B) mRNA: 5′-GCGACTGGAGAGCTGTTTCT and 5′-AACCACATCCTAAGGCCAGC; for beta-ACTIN: 5′-ATGCTCCCCGGGCTGTAT and 5′-CATAGGAGTCCTTCTGACCATTC; and for GAPDH: 5′-TGTGTCCGTCGTGGATCTGA and 5′-CCTGCTTCACCACCTTCTTGA. (Taqman-probes were then created for LC3, beta-ACTIN (5′-CCTAGGCACCAGGGTGTGATG) and GAPDH (5′-CCGCCTGGAGAAACCTGCCAAGTATG) and combined with the PrimeTime Gene Expression Master Mix from Thermo Fischer Scientific. For other mRNA, we used primer and probe sets from Thermo Fischer Scientific: Mm00465434_m1 (mPKD1; spanning exon 1–2), Mm00435829_m1 (mPKD2; spanning exon 1–2), Mm01187303_m1 (mATG5), Mm00503201_m1 (mATG12), Mm01265461_m1 (mBECN1), Mm00448091_m1 (mSQSTM1) and mHif1a (Mm00468869_m1). qPCR was performed with the corresponding StepOnePlus Real-Time PCR System Software and analyzed with the 2^−ΔΔCt method [50 (link)].