Trizol total rna isolation reagent

Cells were either treated or not with native LDL or Mox-LDL at a final concentration of 100 μg/mL for 24 hours. Total RNA was extracted from cells using TRIzol total RNA isolation reagent (Roche Applied Science) and reverse-transcribed using Reverse Transcriptase Core Kit (Eurogentec) according to the manufacturer's instructions. qRT-PCR was performed in 96-well plate format using SYBR Green-based detection on a Step-One-Plus machine (Applied Biosystems) with each 20 μL reaction containing ~50 ng cDNA, 0.3 μM sense and antisense primers, and ABsolute qPCR SYBR Green ROX Mix (Thermo Scientific). The plate was sealed and cycled under the following conditions: 95°C for 15 min, 40 cycles of 95°C for 15 s, 60°C for 30 s, and 72°C for 30 s. Each reaction was performed in triplicate and mRNA levels of RPL27 were used for normalization. The relative expression levels of mRNAs were calculated using the comparative ΔΔCt  method. PCR primers used for the quantification of HO-1 and RPL27 were as follows: 

HO-1 forward primer 5′-AAGACTGCGTTCCTGCTCAAC-3′;

 

HO-1 reverse primer 5′-AAAGCCCTACAGCAACTGTCG-3′;

 

RPL27 forward primer 5′-ATCGCCAAGAGATCAAAGATAA-3′;

 

RPL27 reverse primer 5′-TCTGAAGACATCCTTATGACG-3′.

Cells were either treated or not with native LDL or Mox-LDL at a final concentration of 100 μg/mL for 24 hours. Total RNA was extracted from cells using TRIzol total RNA isolation reagent (Roche Applied Science) and reverse-transcribed using Reverse Transcriptase Core Kit (Eurogentec) according to the manufacturer's instructions. For expression analysis of 94 preselected genes involved in angiogenesis, Human Angiogenesis 96-well StellARray qPCR array (Lonza Ltd., Switzerland) was used according to manufacturer's instruction with ABsolute qPCR SYBR Green ROX Mix (Thermo Scientific) on an ABI Prism 7900HT sequence detection system (Applied Biosystems). Data were analyzed with Global Pattern Recognition Data Analysis Tool (Bar Harbor Biotechnology, Trenton, ME, USA) using the internal array control housekeeping gene expression for normalization.

Cells were either treated or not with native LDL or Mox-LDL at a final concentration of 100 μg/mL for 24 hours and total RNA was extracted from cells using TRIzol total RNA isolation reagent (Roche Applied Science). According to the manufacturer's instructions, The TaqMan microRNA reverse transcription kit (Applied Biosystems) was used to perform gene-specific reverse transcription for each microrna using 10 ng of purified total RNA, 100 mm dNTPs, 50 units of MultiScribe reverse transcriptase, 20 units of RNase inhibitor, and 50 nm gene-specific RT primer samples. Real-time PCRs were carried out on an ABI Prism 7900HT sequence detection system (Applied Biosystems) using 5 μL of the RT product, 10 μL of TaqMan 2x universal PCR master mix (Applied Biosystems), 1 μL of TaqMan microRNA assay mix (containing PCR primers and TaqMan probes) according to the manufacturer's instructions. All quantitative RT-PCRs were performed in triplicate and the relative expression levels of miRNAs were calculated by the SDS 2.3 software (Applied Biosystems) using the comparative ΔΔCt method.