One step rt ddpcr advanced kit for probe

RNA was extracted from RNP-treated cells by using a RNeasy Mini Kit (Qiagen), and the concentration of RNA was determined using a NanoDrop spectrophotometer (Thermo Fisher Scientific). The change in CDKN1A expression was determined by using the One-Step RT-ddPCR Advanced Kit for Probes (Bio-Rad, catalog 1864021) in accordance with the manufacturer’s protocol, with the CDKN1A primers/probe (Bio-Rad, assay 10031252, assay ID: dHSACPE5052298) and with the RPP30 primers/probe (Bio-Rad, assay 10031255, assay ID: dHSAcpe5038241) as controls. Briefly, droplets were prepared using an Automated Droplet Generator (Bio-Rad), and PCR amplification was performed as follows: a first step at 50°C for 60 minutes, a second step at 95°C for 10 minutes, a third step at 95°C for 30 seconds followed by 55°C for 1 minute (for 40 cycles), with a final step at 98°C for 10 minutes. Droplets were analyzed as described above.

Digital PCR was done on a QX200 instrument (Bio-Rad) using the One-Step RT-ddPCR Advanced Kit for Probes (Bio-Rad #1864022) according to the manufacturer’s instructions. Briefly, 22 µl pre-reactions were prepared consisting of 5 µl 4 × supermix, 2 µl reverse transcriptase, 6 µl positive control RNA (125 RNA copies/µl), 15 mM dithiothreitol, 900 nM of each forward and reverse primer and 250 nM E gene hydrolysis probe (FAM) (see higher). 20 µl of the pre-reaction was used for droplet generation using the QX200 Droplet Generator, followed by careful transfer to a 96-well PCR plate for thermocycling: 60 min 46 °C reverse transcription, 10 min 95 °C enzyme activation, 40 cycles of 30 s denaturation at 95 °C and 1 min annealing/extension at 59 °C, and finally 10 min 98 °C enzyme deactivation. Droplets were analyzed by the QX200 Droplet Reader and QuantaSoft software. With an RNA input of 7500 copies per reaction, the digital PCR result was 1500 cDNA copies (or 20% of the expected number, a fraction confirmed by Dr. Jim Huggett for particular lot numbers of #102024, personal communication. The reason for this discrepancy is two-fold: the number of RNA molecules provided by the manufacturer is only approximate, and the reverse transcription reaction is inefficient). The median Cq value of the positive control RNA of 24.55 thus corresponds to 1500 digital PCR calibrated cDNA molecules.

Cell-associated HIV-1 RNA was quantified in peripheral CD4⁺ T cells by ddPCR (One-Step RT-ddPCR Advanced Kit for Probes, BioRad) from samples taken before (0 h) and at the end of each RMD infusion (4 h), at 8 and 24 h (+1 day) after RMD₁, and 72 h (+3 days) and 7 days after RMD₁₋₂₋₃. CA HIV-1 RNA was quantified using two different primers/probe sets annealing to the 5′LTR and GAG conserved regions of HIV-1, to circumvent potential primer mismatch in individuals' viral sequence as previously described (37 (link)). HIV-1 transcription levels were normalized to the housekeeping gene TATA-binding protein (TBP) and shown as relative to levels before RMD₁.

Cells from each condition were collected and RNA was isolated by RNeasy mini kit (Qiagen, Catalog#74104). RNA quality was checked by high sensitivity RNA ScreenTape assay (Agilent, 4200). RNA quantity was determined by Qubit RNA RS kit (Thermo Fisher). RNA samples were mixed with one step RT-ddPCR advanced kit for probes (Bio-Rad), together with ddPCR GEX primer/probe for CD19 or CD22 (Bio-Rad) in a 96 well plate to generate RT-PCR reaction mix. Reaction droplets were generated by QX200 AutoDG droplet generator, PCR reaction was performed by C1000 Touch Cycler (Bio-Rad). RT-PCR droplets were read by QX200 droplet digital PCR system and data was analyzed using Quantasoft (Bio-Rad).

Briefly, total viral RNA was isolated from cell culture supernatant using MagNA Pure LC RNA Isolation Kit-High Performance (Roche Diagnostics Co, Indianapolis, IN, USA) into the MagNA Pure LC 2.0 Instrument automated equipment (Roche Molecular System, Inc, Indianapolis, IN, USA). Quantitative RT-PCR was performed using SARS-CoV-2 N2 gene detection probe (CDC laboratory test kit for SARS-CoV-2) and One-Step RT-ddPCR Advanced Kit for Probes (Bio-Rad) on the QX ONE digital droplet PCR system. The number of viral particles were obtained using QX ONE standard software 1.0 (Bio-Rad, Inc, Irvine, CA, USA). The detection threshold and positive samples were compared by negative and positive viral control.