M mlv first strand synthesis system

Total RNA was extracted from the macrophages cells using Trizol reagent (Invitrogen, Carlsbad, CA, USA). RNA was reverse transcribed using a 5-minute 65 °C incubation of 1 μg total RNA with deoxyribonucleotide triphosphates (Invitrogen) and random primers (Invitrogen). c-DNA was synthesized using an M-MLV First-Strand Synthesis system (Invitrogen), and used for quantitative analysis of mouse genes (supplemental data) with an SYBR Green Master Mix (Applied Biosystems, Foster City, CA, USA). Levels of gene expression were determined by normalization to murine glyceraldehyde 3-phosphate dehydrogenase (GAPDH).

Total RNA was extracted from the macrophages and VSMCs using Trizol reagent (Invitrogen) according to the manufacturer’s protocol. RNA was reverse transcribed using a 5-minute 65 °C incubation of 1 µg total RNA with deoxyribonucleotide triphosphates (Invitrogen) and random primers (Invitrogen). c-DNA was synthesized using an M-MLV First-Strand Synthesis system (Invitrogen), and used for quantitative analysis of mouse genes (Supplementary Table 1) with an SYBR Green Master Mix (Applied Biosystems, Foster City, CA, USA). The relative mRNA expression levels were determined by normalization to murine glyceraldehyde 3-phosphate dehydrogenase (GAPDH) using 2[−ΔΔC(T)] method.

RNAs were isolated using Tri Reagent (Sigma) according to the manufacturer's protocol. 1 μg of RNA was reverse transcribed using M-MLV first-strand synthesis system according to the manufacturer's instructions (Invitrogen) in a total of 20 μl. One microlitre of cDNA preparation was subsequently used in a semi-quantitative PCR analysis according to standard protocol (ReddyMix, Thermo Scientific). PCR amplification was carried out for 20–35 cycles within the linear range of amplification for each gene. PCR products were resolved on 1% agarose or 5% non-denaturing polyacrylamide (for splicing) gels, BET stained and quantified with ImageJ software. The ratios of exon inclusion/exclusion were quantified as a percentage of inclusion/exclusion relative to total intensity of isoform signals. To quantify the mRNA expression, real-time PCR was performed using a Lightcycler 480 (Roche). Reactions were performed with SYBR Green kit (Roche) according to the manufacturer's instructions. PCR cycles were a 15-min denaturation step followed by 50 cycles with a 94 °C denaturation for 15 s, 58 °C annealing for 20 s and 72 °C extension for 20 s. Mouse Rrlp0 mRNA or zebrafish elfa (elongation factor alpha) mRNA were used as standard. Data were analysed with the Lightcycler 480 analysis software. PCR primer sequences are listed in Supplementary Table S1.