Primer probe mix

The SC laboratory used TaqPath ProAmp (TP; ThermoFisher Scientific, Waltham, MA, USA) or FastTaq Universal (FT; ThermoFisher Scientific) for initial testing, since the SC O150 assay had initially been optimized using TP, and all the other assays with FT. Each sample was run in triplicate 7μL reactions (TP) or 10μL reactions (FT). For all TP reactions, a 7μL reaction volume contained 3.5μL of TP, 150nM forward primer, 1000nM reverse primer, 125nM probe, 0.608μL of nuclease-free water, and 2μL of template. Each FT reaction contained 5μL FT, 1μL primer/probe mix (IDT; Integrated DNA Technologies, Inc.), 3μL of nuclease-free water, and 1 μL of template. Smith College used the Applied Biosystems StepOnePlus Real-Time PCR System (ThermoFisher Scientific). Cycling conditions for TP assays had an initial two-minute incubation at 50°C, followed by a 10 min incubation at 95°C and then 40 cycles of 1) 15 sec denaturation at 95°C and 2) one min annealing and extension at 59°C for the SC O150 assay and at 60°C for all other assays. FT assays had an initial 30 sec incubation at 60°C, followed by a one min incubation at 95°C and 40 cycles of 1) one sec denaturation at 95°C and 2) 20 second annealing and extension at 60°C.

For cDNA synthesis and qPCR, RNA was quantified using a Nanodrop instrument (Thermo Fisher), and 100 ng was loaded into the iScript cDNA Synthesis Kit (1708891, Bio-Rad, Hercules, CA). For IL21 gene analysis, a predesigned primer probe mix was purchased from Integrated DNA Technologies (Assay ID: Mm.PT.58.7853071, Integrated DNA Technologies, San Jose, CA) and normalized using ACTB as a housekeeping gene (Assay ID: Mm.PT.39a.22214843.g). cDNA was assayed using the Luna Universal Probe qPCR master-mix (M3004X, New England Biolabs, Ipswich, MA), on an Applied Biosystems StepOne Real-Time PCR System (Applied Biosystems, Foster City, CA). Relative quantitation was performed using the delta-delta CT method, described previously (55 (link)).