Rotor gene qpcr probe kit

Culture supernatants were collected from infected MC, MDM^EI or MDM^LI 7-days post infection unless otherwise specified. Supernatants were first spun to remove cellular debris, filtered using 0.22 μm filter and frozen at -80 ⁰C until use. RNA was extracted from 140 μl of culture supernatant using the Qiagen mini-Viral Prep kit according to the manufacturer's protocol. RNA (~3 μg) was treated with 2 units turbo-DNase (Invitrogen) in 1X turbo-DNase buffer for 30 min at 37°C, to remove DNA carryover and then purified using Qiagen RNEasy kit. About 1.3 μg of RNA was used to generated cDNA with the Qiagen OmniScript cDNA preparation kit following the manufacturers protocol. Using the cDNA as input material and the Rotor Gene qPCR probe kit (Qiagen), the genomic HIV RNA copies in the supernatants of infected MC and MDM cultures were measured relative to a standard curve developed with the J-lat HIV integration standard cell line (see above). Thermal cycling conditions for qPCR consisted of 95°C for 5 min, and 40 cycles of denaturation at 95°C for 10 sec and annealing and extension at 60°C for 30 sec. The measurements are reported as RNA copies per provirus present.

RNA was extracted from AM, MDM or HAP1 cells using the RNeasy Mini Kit (Qiagen) following the manufacturer's protocol. About 500 ng of RNA was used to synthesize cDNA with High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems, #4368813) following the manufacturers protocol. The cDNA was used as input material and the Rotor Gene qPCR probe kit (Qiagen, #204374), mRNA copies of infected AM and MDM or uninfected controls were measured and normalized to an internal control gene (18S rRNA). Thermal cycling conditions for qPCR consisted of 95 °C for 5 min, and 40 cycles of denaturation at 95 °C for 10 s and annealing and extension at 60 °C for 30 s. The relative expression levels were calculated using the 2^−ΔΔCT method, where the ΔΔC_t is the difference in ΔC_t between the target and reference samples: $$\mathrm{\Delta }\mathrm{\Delta }{\text{C}}_{\text{t}}=\mathrm{\Delta }{\text{C}}_{\text{t},\text{AM}/\text{MDM}}-\mathrm{\Delta }{\text{C}}_{\text{t},\text{HAP1}}={\left({\text{C}}_{\text{t},\text{UBER}}-{\text{C}}_{\text{t},\text{18S}}\right)}_{\text{AM}/\text{MDM}}-{\left({\text{C}}_{\text{t},\text{UBER}}-{\text{C}}_{\text{t},\text{18S}}\right)}_{\text{HAP1}}.$$