Tri reagent rna isolation reagent

The efficiency of ITGB1 siRNA transfection was assessed with mRNA expression using Real-Time Quantitative PCR (qPCR) assay. Total RNA from cultured ASM cells was extracted using the TRI Reagent RNA Isolation Reagent (#T9424, Sigma). Total RNA weighing 500 ng was applied to generate 1st strand cDNA by using the Revert Aid First Strand cDNA Synthesis Kit (#K1622, Thermo, MA). The sequences of associated qPCR primers for rat ITGB1 were derived from the previous report including GAA​TGG​AGT​GAA​TGG​GAC​AGG​AG (ITGB1 forward), CAG​ATG​AAC​TGA​AGG​ACC​ACC​TC (ITGB1 reverse), and the control GAPDH primers AGG​TCG​GTG​TGA​ACG​GAT​TTG (forward) and GGG​GTC​GTT​GAT​GGC​AAC​A (reverse) (Luo et al., 2018 (link)). The primers were synthesized from General Biosystems (Anhui, China), and PowerUp SYBR Green Master Mix (#A25742, Applied Biosystems, CA) was used for PCR amplification. The reaction was run in the qRT-PCR system (StepOnePlus, Applied Biosystems) with 1 µL of the cDNA in a 10 µL reaction according to the manufacturer’s instructions. Calibration and normalization were done using the 2^−∆∆CT method, where ∆∆CT = CT (target gene) -CT (reference gene), and CT referred to the PCR cycle number by reaching the defined fluorescence intensity. Fold changes in mRNA expression were calculated based on the resulting CT values from three independent experiments.

Total cellular RNA was isolated using TRI^® Reagent RNA Isolation Reagent (Sigma Aldrich) from snap frozen liver tissue following the instructions of the manufacturer. Complementary DNA (cDNA) was generated from 2 µg of total RNA using the High Capacity cDNA Reverse Transcription Kit (Applied Biosystems) following the instructions of the manufacturer. iQ™ SYBR^® Green Supermix (BioRad) was used for quantitative PCR (qPCR). Primer sequences were obtained from the qPrimerDepot (http://mouseprimerdepot.nci.nih.gov) and will be provided upon request. Melting curve analysis and agarose gel electrophoresis was performed to assess the quality of primers and the qPCR.

Total RNA was isolated from carotid artery homogenates using TRI^® Reagent RNA Isolation Reagent (Sigma-Aldrich) and retro transcribed into cDNA using iScript^™ Select cDNA Synthesis Kit (Bio-Rad). Real-time PCR was carried out with a C1000^™ Thermal Cycler (Bio-Rad) using SYBR Green (Bio-Rad). TNF-α, IL-6, and Ariginase-1 (Arg1) were normalized against GAPDH. Primers used for RT-PCR were as follows:
TNF- α: sense primer, 5′-CACAGAAAGCATGATCCGCGACGT-3′;
Antisense primer, 5′-CGGCAGAGAGGAGGTTGACTTTCT-3′;
IL-6: sense primer, 5′-TCCAGTTGCCTTCTTGGGAC-3′;
Antisense primer, 5′-GTACTCCAGAAGACCAGAGG-3′;
Arg1: sense primer, 5′-CTCCAAGCCAAAGTCCTTAGAG-3′;
Antisense primer, 5′-AGGAGCTGTCATTAGGGACATC-3′;
GAPDH: sense primer, 5′-AACGACCCCTTCATTGAC-3′;
Antisense primer, 5′-TCCACGACATACTCAGCAC-3′;
Final data are presented as relative fold change against sham.

RNA extraction from fruit at the ‘ripe’ stage was performed of approximately 200 mg powder tissue with TRI Reagent RNA isolation reagent (Sigma‐Aldrich) according to the manufacturer’s protocol. Reverse transcription and DNase treatment were performed with the iScript™ gDNA Clear cDNA Synthesis Kit #172‐5035 (Bio‐Rad). To eliminate genomic DNA contamination, the cDNA was amplified using ACTIN primers (Table S3) that differentiate between genomic DNA and cDNA sequences. The cDNA was amplified by ProFlex PCR System (Applied Biosystems by Thermo Fisher Scientific). Quantitative polymerase chain reactions were performed using the Applied Biosystems™ Fast SYBR™ Green Master Mix on a StepOnePlus™ Real‐Time PCR System (Applied Biosystems). Cycling conditions were 95 °C for 20 s, followed by 40 cycles of 95 °C for 3 s, 60 °C for 30 s and fluorescence acquisition at 60 °C. For each gene, the relative mRNA level was determined in three biological replicates. The gene ACTIN served as a control for normalization. Primers used for RT‐PCR amplification are described in Table S3.

Total RNA was extracted using TRI Reagent RNA Isolation Reagent (Sigma) according to the manufacturer's protocol. Reverse transcription was performed using the iScript cDNA Synthesis Kit (Bio-Rad Laboratories, Inc., Hercules, CA, USA) with 1 μg of total RNA for reaction. Gene-specific primers and probes were used for real-time qPCR. PCR amplification and fluorescence detection were performed using a Light Cycler 480 Real-Time PCR detection system (Roche), and the threshold cycles were determined using Light Cycler 480 Software. Fold inductions were determined using the ΔΔCt method against the GAPDH gene.

For gene expression profiling, we implemented a method for purification of intact pancreatic RNA according to a modified guanidinium salts method (MacDonald et al, 1987). Briefly, a small piece of pancreatic tissue was immediately homogenized with a Polytron (VWR) in pre‐cooled 4 M guanidinium thiocyanate with 112 mM beta‐mercaptoethanol, centrifuged at 5,000 g for 5 min at 4°C to remove insoluble debris. RNA was precipitated from the supernatant with pre‐cooled 75% ethanol, 0.1M potassium acetate, pH 5.5, and 75 mM acetic acid at −20°C for 2 h. The precipitate was pelleted by centrifugation at 10,000 g for 10 min at 4°C and resuspended at room temperature in 7.5 M guanidinium HCl and 10.5 mM beta‐mercaptoethanol. The RNA was re‐precipitated twice with 0.1 M potassium acetate, pH 5.5, and 50% ethanol to remove residual RNases, followed by purification with TRI Reagent RNA Isolation Reagent (Sigma‐Aldrich). The concentration and quality of RNA was measured with a NanoDrop spectrophotometer (ND‐1000, Thermo Scientific) and an Agilent 2100 Bioanalyzer. RNA integrity numbers ranged from 7.8 to 9.3.
Alternatively, we used a previously described protocol (Cobo et al, 2018).