Onestep plus

Physical titers of HIV and MLV vector supernatants were determined by a qPCR-based product-enhanced RT (PERT) assay similarly as described previously.⁷⁹ (link)^,80 (link) Briefly, 5 μL of 1:100 diluted plain vector supernatant were mixed with 5 μL 2× lysis buffer (100 mM Tris [pH 7.4], 50 mM KCl, 0.25% [v/v] Triton X-100, 40% [v/v] Glycerol; 400 U/mL RiboLock RNase inhibitor added prior to use) and incubated for 20 min at room temperature. Subsequently, the sample was diluted 1:5 by addition of 40 μL ddH₂O. For qPCR amplification, 5 μL of diluted sample or standard was mixed in duplicates with 20 μL PERT qPCR mix composed of 2.79 nM mM MS2 RNA (Roche), 0.5 μM fwd (5′-TGCTCGCGGATACCCG-3′) and rev (5′-AACTTGCGTTCTCGAGCGAT-3′) MS2 primer, 0.25 μM MS2 probe (5′ HEX-ACCTCGGGTTTCCGTCTTGCTCGT-BHQ2 3′), 0.5 μM ROX reference dye, and 1.25× Maxima Probe Master Mix (ThermoScientific). Amplifications were performed on a OneStepPlus (Applied Biosystems) with one cycle at 42°C for 20 min, 95°C for 2 min for reverse transcription, followed by 40 cycles of 95°C for 30 s, 58°C for 30 s, and 72°C for 30 s for amplification. As standard 5 μL of a 10-fold dilution series of recombinant HIV-1 RT (Abcam #ab63979; 2 mU/μL in DMEM, 10% FCS) was used in the range of 10⁵–10⁰ nU RT/reaction. Infectious and physical titers of vector supernatants employed in this study are summarized in Table S1.

The NTS was mircropunched using anatomical landmarks, and NG from the same animal were pooled before mRNA was extracted using Trizol (Life Technologies). RT quantitative PCR was performed in triplicate using SybR Green on a OneStep Plus instrument (Applied Biosystems), and results were analyzed using the 2ddCt method (Livak and Schmittgen, 2001 (link)).