RT-qPCR Protocol for Human Lung Fibroblasts

cDNA was synthesized from RNA extracted from human lung fibroblasts using the iScript cDNA Synthesis Kit (Cat# 1708891, Bio-Rad, Hercules, CA, USA) according to the manufacturer’s instructions. We used 1 μg of RNA per 20 μL cDNA reaction. cDNA was synthesized on C1000 Touch Thermal Cycler (Bio-Rad, Hercules, CA, USA) with the following protocol: A. Priming stage for 5 min at 25 °C. B. Reverse Transcription stage for 20 min at 46 °C. C. RT inactivation stage for 1 min at 95 °C. D. Hold at 4 °C.
The following reaction mixes were used for qPCR: A. 5 μL of TaqMan gene expression master mix (Cat# 4369016, ThermoFisher Scientific, Waltham, MA, USA) B. 3.5 μL of UltraPure DNase/RNase-Free Distilled Water (Cat# 10977015, ThermoFisher Scientific, Waltham, MA, USA) C. 0.5 μL of GAPDH and B2M for duplexed housekeeping gene controls or D. 0.5 μL target FAM primer. One μL of cDNA was added to 9 μL of the aforementioned primer reaction mix. The reaction was performed on the StepOne Plus Real-time PCR machine by Applied Biosystems (ThermoFisher Scientific, Waltham, MA, USA) using the following protocol: Holding stage (1) 15 min at 48 °C, (2) 10 min at 95 °C. Cycling Stage (1) 1 min at 95 °C, (2) 1 min at 60 °C for a total of 40 cycles. The target gene 2^−ΔCT values normalized to GAPDH or B2M were graphed and statistically analyzed in GraphPad Prism version 9 (GraphPad Software Inc., La Jolla, CA, USA). All qPCR primers utilized are listed in Table S5.