Mastercycler gradient pcr thermal cycler

RNA was isolated from the samples (200 ng/μl) using the Ambion by Life Technologies Purelink RNA minikit (Thermo Fisher Scientific, MA), following the manufacturer’s instructions. cDNA was made following the instructions on the Applied Biosystems high-capacity cDNA reverse transcription kit (Thermo-Fisher Scientific, MA), and PCR was conducted using a Master Cycler gradient PCR thermal cycler (Eppendorf, NY). Using GAPDH (glyceraldehyde-3-phosphate dehydrogenase) as the housekeeping gene, qPCR was set up with each sample being run on the plate in triplicate for each of the conditions. A list of the primer sequences used can be found in Table S1 in the supplemental material. Power SYBR green PCR master mix was used (Thermo Fisher Scientific, MA).

The myofibroblast-populated collagen RNA extraction protocol used in this study is different from those in earlier studies as it is optimized for higher protein concentrations. After incubation and aspiration of media, the collagen matrix was collected in microcentrifuge tubes for high-speed centrifugation for 5 min, and then the supernatant was discarded. An aliquot of 100 μl prewarmed 0.25 mg/ml collagenase was added to each tube and incubated for 15 min at 37°C. RNA was isolated from the samples using the Direct-zol RNA miniprep kit (Zymo Research, CA) following the manufacturer’s instructions, and TRIzol reagent was used to lyse the samples. The RNA concentration was measured using a NanoDrop ND-1000 (Thermo Scientific). The cDNA was made following the instructions on the Applied Biosystems high-capacity cDNA reverse transcription kit (Thermo-Fisher Scientific), and PCR was conducted using a MasterCycler gradient PCR thermal cycler (Eppendorf, NY). Quantitative PCR was set up with each sample being run on the plate in triplicate for each of the conditions, as described earlier using GAPDH as the optimized reference gene. A list of the primers used can be found in Table S1.