Fig. 4

Amplification plot of a known concentration of GAPDH (gBlock gene fragment; Integrated DNA Technologies) ranging from 0.2 μg to 0.0002 μg (or 200 pg) in a 1:4 serial dilution. The quantitative PCR reaction was performed in a QuantStudio 6 Flex Real-Time PCR System (Applied Biosystems) using FAM as the reporter dye, NFQ-MGB as the quencher, and ROX as the passive reference. The RT-qPCR assay was performed using a pre-designed PrimeTime® Mini qPCR Probe from IDT Integrated DNA Technologies, which consists of a GAPDH primer pair and 5′ nuclease probe. The RT-qPCR reaction was performed using 10 μL of qPCR mixture containing 4 μL cDNA template, 5 μL of the PrimeTime gene expression master mix (Cat# 1055770, IDT Integrated DNA Technologies), 0.5 μL of PrimeTime®qPCR Assay (Premixed primers and probe), 0.5 μL nuclease-free water in a MicroAmp Optical 384-well reaction plate (Applied Biosystems). The qPCR reactions were performed using the following conditions: 3 min at 95 °C, 40 cycles of 15 s at 95 °C, and 1 min annealing at 60 °C. Actual Cts, standard curve dilutions, and GAPDH concentrations are presented in Suppl. Materials Table 1