Gotaq qpcr master mix with bryte green

RNA was extracted and purified from cultured and uncultured cells using the ReliaPrep RNA Cell Miniprep System (Promega, Madison, WI, USA). Total RNA was converted to cDNA by reverse transcription using the Revert Aid Kit (Thermo Fisher Scientific, St. Leon-Rot, Germany). Quantitative real-time polymerase chain reaction was performed using the GoTaq qPCR Master Mix with BRYTE green (Promega). A 7500 real-time PCR system was used and data was analyzed using System SDS software v2.0.6 (Applied Biosystems). Fold change differences between samples were determined using the comparative Ct method (∆∆Ct). The expression level of different target genes, relative to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and the calibrator, was given by 2-∆∆Ct. Gene-specific primers were produced by Integrated DNA Technologies (Leuven, Belgium).

Total RNA from cultured cells was extracted using ReliaPrep RNA Cell Miniprep System (Promega, Madison, WI, USA). RNA was reverse-transcribed into complementary DNA using the Revert Aid kit (Thermofisher) and performed at 25 °C for 10 min, 30 min at 50 °C. For quantitative real-time polymerase chain reaction (qPCR), GoTaq qPCR Master Mix with BRYTE green (Promega) was used and the samples were subsequently subjected to qPCR in an ABI 7500 real-time PCR system and analyzed using System SDS software (Applied Biosystems). For analysis according to the comparative Ct (δδCt) method, each Ct value was first normalized to the house-keeping gene Gapdh. Gene-specific primers were produced by Integrated DNA Technologies (Suppl. table 1).

Slices were snap frozen in liquid nitrogen and stored at −80°C until RNA extraction. Total RNA was extracted with TRIzol Reagent (Thermofisher) according to the manufacturer’s manual. Samples were homogenized by crushing the tissue with 5 mm steel beads (Qiagen) with a Retsch MM 400 laboratory mill at 30 Hz for 2 min. Afterward, RNA was reverse transcribed into cDNA using the MLV Reverse Transcriptase (Promega). For qPCR, GoTaq qPCR Master Mix with BRYTE green (Promega) was used. qPCR was done using the Quantstudio3 real-time PCR system (Thermofisher). Gene-specific primers (Table 2) were produced by Integrated DNA Technologies (IDT Leuven). For analysis according to the Delta–Delta threshold (Ct) method, each Ct value was normalized against the respective reference genes (for mouse: the mean of Gapdh and Gtf2b; for human: the mean of GAPDH and YWHAZ). The best reference genes were selected out of six genes using GeNORM, performed in R environment. Each data point corresponds to one PCLS.