Sybr green qrt pcr

HUVECs were transduced with V-miR-1 (or control, V-ctrl) and subjected to Argonaute-2 RNA immunoprecipitation assay followed by small sequencing, as described previously (15 (link)). Validation of mRNA levels in input and immunoprecipitate samples was performed using SYBR Green qRT-PCR (Bio-Rad).

Total RNA was extracted using the TRIzol method (Life Technologies, United States). First-strand cDNA was prepared using a reverse-transcription kit (Life Technologies, United States). SYBR Green qRT-PCR was performed to measure the gene expression on a qPCR platform (Bio-Rad, United States) with specific primer pairs as follows: HIF-1α (forward: 5′-TGATGTGGGTGCTGGTGTC-3′, reverse: 5′-TTGTGTTGG GGCAGTACTG-3′), CCL2 (forward: 5′-AGGTCCCTGTCAT GCTTCTGG-3′, reverse: 5′-CTGCTGCTGGTGATCCTCTTG- 3′), PECAM1 (forward: 5′-CCAAAGCCAGTAGCATCATGG TC-3′, reverse: 5′-GGATGGTGAAGTTGGCTACAGG-3′), IF N-γ (forward: 5′-CAGCAACAGCAAGGCGAAAAAGG-3′, re verse: 5′-TTTCCGCTTCCTGAGGCTGGATα-3′), TGF-β (for ward: 5′-ACTGATACGCCTGAGTGGCT-3′, reverse: 5′-CCCT GTATTCCGTCTCCTTG-3′), and β-actin (forward: 5′-AAGG CCAACCGTGAAAAGAT-3′, reverse: 5′-GTGGTACGACCAG AGGCATAC-3′) as control.

Total RNA was isolated from neutrophils using TRIZOL reagent (Invitrogen) according to the manufacturer’s instructions. Genomic DNA contamination was removed by treating total RNA with DNaseI (Thermo Fisher Scientific) prior to conversion to cDNA using a RevertAid First-strand cDNA Synthesis Kit (Thermo Fisher Scientific, Cat. No: K1622, Waltham, MA). SYBR green qRT-PCR (BioRad, California, USA) was performed as mentioned elsewhere with some modifications (Kobpornchai et al., 2020 (link)). The reactions were amplified using specific primers for human cytokines and chemokines including IL-1β, IFN-γ, TNF-α, IL-6, IL-8 and CCL3 (Supplementary Data 1: Table S1). GAPDH was used as housekeeping gene. The amplification was performed with Eppendorf Realtime PCR (Realplex4, Eppendorf, Hamburg, Germany), consisted of a first denaturation step at 95°C for 5 min, followed by 40 cycles at 95°C for 15 s and 60°C for 60 s, and a melting curve step at 65–95°C. Relative mRNA expression was calculated using the comparative Ct method with the formula 2^-ΔΔCt.