Figure 2.

High cell lysate concentrations were shown to be inhibitory to RT-qPCR, thus modifications to current RT-qPCR conditions were required. (a) The lysate concentration of a single cell encapsulated in a 1 nL droplet is equivalent to approximately 1000 cells/μL on the scale of conventional RT-qPCR volumes. (b,c) To demonstrate the inhibitory effect, one-step RT-qPCR was performed to detect Gata3 expression in EL4 cells at 10 and 1000 cells/µL concentration in a 10 µL reaction volume, as well as the amount of total RNA equivalent to 1000 cells/μL (10 ng RNA/μL). Inhibition was observed as either a C_T delay or sporadic fluorescence generation with no exponential phase at 1000 cells/µL (b). Gel electrophoresis also showed reduced amplification products at higher cell concentration (c). See Supplementary Figure S2 for full-length gel image. (d) Gata3 mRNA was quantified efficiently on a range of 1 to 10⁴ EL4 cell(s) (shown by darkest to lightest colour plots) in a 10 µL RT-qPCR using our in-house one-step RT-PCR mix with gp2.5. (e) Dilutions of cells or (f) Gata3 and EPCR IVT doped in constant cell lysate concentrations (1000 cells/µL) were analyzed by RT-qPCR and plotted against their C_T values to demonstrate high amplification efficiencies using our in-house one-step RT-PCR mix. (g) To emulate the range of mRNA expected from single cells, IL-7Rα, Gata3, and EPCR IVT were doped in constant cell lysate concentrations (1,000 cells/µL) at either 1 or 10,000 copies/nL. (h) Using our in-house, multiplex, one-step RT-PCR mix, the three target genes were simultaneously quantified in four different combinations of transcript levels. The difference in C_T values obtained using our single and triplex assays were found to be insignificant after 40 thermal cycles (P > 0.1). Each dot represents one technical replicate. Data was analyzed and plotted using JMP (version 15.2.1 www.jmp.com)