Rt2 sybr green mastermix

AECs and airway-derived fibroblasts were grown in 6-well plates to 80% confluence, at which point RNA was collected using RNeasy Mini Kits (Qiagen). 500 ng of RNA was used to synthesize cDNA using the RT² First Strand Kit (Qiagen). cDNA was then combined with 2× RT2 SYBR Green Mastermix (Qiagen) and RNase-free water and distributed onto a manufacturer optimized 384-well Human Epigenetic Chromatin Modification Enzymes Focused Array (PAHS-085E-4, Qiagen) pre-loaded with primers targeting 84 genes encoding epigenetic enzymes and 5 housekeeping genes as per manufacturer’s protocol. A complete list of the genes that were analyzed is available in Table S1 (Additional file 1). Additionally, to identify gene expression of CREBBP and EP300, cDNA was combined with 2× RT2 SYBR Green Mastermix, RNase-free water, and primers targeting CREBBP (PPH00324F-200, Qiagen), EP300 (PPH00319A-200, Qiagen), hypoxanthine phosphoribosyltransferase 1 (HPRT1, PPH01018C, Qiagen), ribosomal protein L13a (RPL13A, PPH01020B-200, Qiagen), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH, PPH00150F, Qiagen) and loaded onto 384-well reaction plates. Data cleaning and housekeeping gene selection is described in Supplementary Methods (Additional file 2). Target gene expression was calculated using the delta Ct method: 2^(C_tHousekeeping Gene – C_tTarget Gene)*10000.

Lung samples were incubated in RNA stabilization reagent for 24 h at 4 °C and stored at −80 °C. Total RNA isolation was performed with an RNAeasy Mini Kit (Qiagen) according to the manufacturer’s instructions. Reverse transcription and complementary DNA (cDNA) synthesis were performed using an RT² First Strand Kit (Qiagen) and 1 μg RNA. Commercially available PCR arrays, including an Angiogenesis Array, were obtained from SA Biosciences (Frederick, MD). The PCR array contained 84 primer pairs that amplify genes involved in rat angiogenesis. Briefly, cDNA was mixed with 2 × RT² SYBR Green Master Mix (1.35 mL; Qiagen) and RNase-free water to a final volume of 2.7 mL. Each well in the RT² Profiler PCR array plate contained 25 μL of sample. PCR was performed on a Takara TP-800 thermal cycler (Takara, Shiga, Japan) following the manufacturer’s instructions. The housekeeping genes hypoxanthine phosphoribosyltransferase 1 (Hprt1) and ribosomal protein lateral stalk subunit P1 (Rplp1) were used to normalize expression levels, calculated using ΔCt values. Fold-changes in expression were calculated using the ΔΔCt (threshold cycle) method. ΔΔCt values in the EC and EC–ASC groups were determined and the fold-changes were calculated as 2^(−ΔΔCt).

The Human Endothelial Cell Biology RT² Profiler^™ PCR Array (Qiagen) was utilized to screen for gene targets by TRAIL (Table S1). Total RNA was harvested from ibidi samples using the mirVana® Isolation kit. Reverse transcription was performed using an RT² PreAMP cDNA Synthesis kit (Qiagen). CDNA was pre‐amplified with RT² PreAMP Pathway Primer mix (Qiagen). PCR reaction mixtures were as follows: 1,350 µl of 2× RT² SYBR Green Master mix (Qiagen), 102 µl of cDNA synthesis reaction, and 1,248 µl of RNase‐free water. A 25 µl of aliquot was added to each well, sealed and centrifuged at 1,000g for 1 min. Amplification of cDNA targets was achieved using the Applied Biosystems 7900HT Fast RT‐PCR system. Samples were denatured at 95°C for 10 min, followed by 40 cycles at 95°C for 15 s and 60°C for 1 min. Cyclic threshold (C_T) values were calculated for each reaction and results were analyzed using a web‐based software (www.SABiosciences.com/pcrarraydataanalysis.php).