Illustra rnaspin rna isolation kit

Total cellular RNA was isolated from cultured bone marrow macrophages using the illustra RNAspin RNA isolation kit, according to the manufacturer’s instructions (GE Healthcare). The RNA content and purity were determined by measuring absorbance at 260/280 nm on NanoDrop One (Thermo Fisher Scientific) and the quality of RNA was determined using TapeStation System (Agilent).

Total RNA was extracted from the aorta and cells using an illustra RNAspin RNA Isolation Kit (GE Healthcare). cDNA was synthesized from 100 ng of total RNA using a QuantiTect Reverse Transcription kit (Qiagen). Quantitative RT-PCR (qPCR) was performed on an Mx3000P (Agilent Technologies) using Power SYBR Green PCR Master Mix (Applied Biosystems). Mouse PCR primers were as follows: VCAM-1, sense 5′-CCCGTCATTGAGGATATTGG-3′ and antisense 5′-GGTCATTGTCACAGCACCAC-3′; ICAM-1, sense 5′-TTCACACTGAATGCCAGCTC-3′ and antisense 5′-GTCTGCTGAGACCCCTCTTG-3′; monocyte chemoattractant protein (MCP)-1, sense 5′-CCACTCACCTGCTGCTACTCAT-3′ and antisense 5′-TGGTGATCCTCTTGTAGCTCTCC-3′; F4/80, sense 5′-TGCATCTAGCAATGGACAGC-3′ and antisense 5′-GCCTTCTGGATCCATTTGAA-3′; and β-actin, sense 5′-CCTGAGCGCAAGTACTCTGTGT-3′ and antisense 5′-GCTGATCCACATCTGCTGGAA-3′. Human PCR primers were as follows: VCAM-1, sense 5′-ATGAATTCGAACCCAAACA-3′ and antisense 5′-CCTGGCTCAAGCATTGTCATA-3′; MCP-1, sense 5′-CCCCAGTCAACCGCTGTTAT-3′ and antisense 5′-AGATCTCCTTGGCCACAATG-3′; ICAM-1, sense 5′-TGATGGGCAGTCAACAGCTA-3′ and antisense 5′-GGGTAAGGTTCTTGCCCACT-3′; and glyceraldehyde 3-phosphate dehydrogenase (GAPDH), sense 5′-TGGGTGTGAACCATGAGAAG-3′ and antisense 5′-GCTAAGCAGTTGGTGGTGC-3′. Data are expressed in arbitrary units that were normalized by β-actin or GAPDH.

HUVECs were cultured in 24-well plates to 100% confluency in Comp-MCDB medium, then treated with IFNγ (20 ng/mL), bradykinin (20 μM), or LPS (100 ng/mL) for 2 or 24 h. RNA isolation was carried out using the illustra™ RNASpin RNA Isolation Kit (GE Healthcare, Chicago, CA, USA) according to the manufacturer’s protocol. RNA–cDNA transcription was performed with the Tetro cDNA Synthesis Kit (Bioline, Essex, UK). SensiFAST SYBR Master Mix—No ROX Kit (Bioline) was used for the quantification of cDNA using a Rotor-Gene Q (Qiagen, Hilden, Germany) real-time PCR cycler. Primers (Table 2) were designed using the NCBI Primer-BLAST primer design tool and synthetized using IDT (Coralville, IA, USA). The purity and size of PCR products were checked by sequencing (sequencing was performed by Biomi Ltd., Gödöllő, Hungary) after the first use of each primer pair and by high-resolution melting curve analysis for each measurement.
Quantification was performed using the Rotor-Gene Q Pure Detection 2.1.0 software (Qiagen, Hilden, Germany), and values of interest were normalized with that of β-actin.