Taqman primer assays

Total RNA containing miRNA was isolated from hPASMC and lung tissue samples using QIAzol lysis reagent and column purified using a miRNeasy kit (Qiagen). After quantification with Nanodrop 2000 spectrophotometer (ThermoScientific, Rockford, IL), miRNAs were reversely transcribed using a RT2 miRNA First Strand Kit (SABiosciences, Frederick, MD). For qRT-PCR analysis of miRNA expression, a poly (A) tail was first added to the 3′-end of miRNAs using a Poly (A) Polymerase Tailing Kit (Epicentre Biotechnologies, Madison, WI). Poly (A) tailed-miRNAs were then reversely transcribed using M-MLV Reverse Transcriptase (Invitrogen, Grand Island, NY) with a poly (T) adaptor, which includes a poly (T) sequence and a sequence complementary to the universal primer used in following qRT-PCR analysis. SNORD44, SNORD47 and SNORD48 were used as internal controls. The expression of mRNAs was determined using specific TaqMan primer assays (Applied Biosystems) with GAPDH used as an internal control. Real-time PCR analysis was performed using a CFX384 system (Bio-Rad), and relative changes in mRNA and miRNA expression were calculated after normalization to their respective internal controls using the comparative Ct method.

Total RNA was isolated from adipose tissue (100 mg) using the RNeasy Lipid Tissue Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer’s protocol. Assessment of purity and quantity was measured with both a spectrophotometer (NanoDrop 1000, Thermo Scientific, Wilmington, DE, USA) and with a Bioanalyzer 2100 (Agilent Technologies, Santa Clara, CA, USA). A starting sample of 300 ng of RNA was reverse transcribed for each case with the iScript kit (Bio-Rad, Hercules, CA, USA). Using TaqMan technology (Applied Biosystems, Waltham, MA, USA), quantitative real-time PCR was performed in triplicate on a 7500 instrument (Applied Biosystems) with the respective controls and quality checks included. Pre-designed exon-exon spanning TaqMan primer assays from Applied Biosystems were used (ID: TNF-α: Hs01113624_g1; SOCS3: Hs02330328_s1) and amplified in singleplex with the housekeeping gene peptidylprolyl isomerase A (PPIA: Hs99999904_m1) [48 (link)]. Gene expression was normalized using the 2−ΔCt method, including correction for amplification efficiency calculated from the standard curves for each primer set [49 (link),50 (link)]. As in previous reports [16 (link),17 (link),18 (link)], gene expression of PPIA showed no group differences in both VAT and SAT.

Representative genes with ≥2 fold differential expression identified in the fibrosis array were validated by real-time PCR TaqMan gene expression assays (Applied Biosystems, Foster City, USA). Gene specific TaqMan primer assays purchased from Applied Biosystems are indicated in S1 Table. cDNA was prepared with High-Capacity cDNA Reverse Transcription Kits (Applied Biosystems) according to the kit protocol starting with 100–200 ng of total RNA. Real-time PCR was performed on the ViiA7 system in a 96-well format. Each sample was measured in triplicate using the TaqMan Gene Expression Master Mix (Applied Biosystems). The cycling program was set as 50°C for 2 mins, 95°C for 10 mins followed by 40 cycles at 95°C for 15 secs and 60°C for 1 min. The average cycle threshold value for housekeeping gene β-actin was used to normalize the raw cycle threshold data and calculate ΔC_t. The ΔΔC_t for each Gene Of Interest (GOI) was calculated by deducting the average ΔC_t of GOI in the control tenons samples from the ΔC_t of each GOI in the AGV capsules. The relative gene expression was reported as fold-change of each GOI compared to the control calculated using the formula 2^-ΔΔCt.