Paxgene rna isolation kit

Blood was withdrawn in PAXgene tubes (PreAnalytix, GmbH, Germany) at baseline, i.e. 5 min before the glucose clamp test, and 70 min after the hyperglycemic clamp of 10 mm/L l for controls or 15 mm/L for T2DM patients and individuals with MetS .
Total RNA was isolated from peripheral blood using the PAXgene RNA isolation kit according to the manufacturers’ instructions including a DNAse (Qiagen, Venlo, The Netherlands) step to remove genomic DNA. RNA samples were further processed by ServiceXS (Leiden, The Netherlands). Amplification was performed using the Ambion® Illumina TotalPrep RNA Amplification Kit (Ambion, # IL1791), resulting in biotinylated, amplified cRNA. Labelled RNA samples were hybridized to Sentrix Human HT12v3 Expression bead chip arrays (Illumina, San Diego, CA). Signal was developed with streptavidin-Cy3 and the BeadChip was scanned with the Illumina BeadArray Reader, which is a two-channel, 0.8 μm resolution confocal laser scanner, followed by feature extraction. Bead summary intensities were log2-transformed and normalized using quantile normalization [19 (link)].

Total RNA was isolated from peripheral blood using the PAXgene RNA isolation kit according to the manufacturers’ instructions including a DNAse (Qiagen, Venlo, The Netherlands) step to remove genomic DNA. RNA samples were concentrated by SpeedVac for 30 min. RNA purity and concentration was measured using NanoDrop ND-1,000 Spectrophotometer (Thermo Scientific, Breda, The Netherlands). cDNA was synthesized from 500 ng total RNA per sample using RevertAid™ H Minus First Strand cDNA Synthesis Kit (Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer’s recommendations. Quantitative real-time polymerase chain reaction (PCR) was performed in an ABI PRISM 7900HT system (Applied Biosystems, Foster City, CA, USA), using primers designed by Primer Express version 2.0 (Applied Biosystems):Briefly, in a 10 μl reaction volume, 4 μl of diluted cDNA, 5 μl SYBR Green PCR Master Mix (Applied Biosystems), and 0.5 uM of each gene-specific primers were mixed. Gene expression levels were calculated using an arbitrary standard curve and normalized to the human housekeeping gene β-actin. Comparisons were performed using Student’s t-test (two-sided). Differences were considered statistically significant if probability values (P) were less than 0.05. Results are presented as mean ± standard error of the mean (SEM).

RNA was isolated from the cell lysates and PAXgene tubes using the RNeasy Micro or Mini kit (QIAGEN Benelux BV) or the PAXgene RNA isolation kit (PreAnalytiX), respectively, according to the manufacturers’ protocols. In both procedures, a DNase (QIAGEN Benelux BV) step was included to remove any genomic DNA. RNA quantity and purity were determined using a NanoDrop spectrophotometer (NanoDrop Technologies, Wilmington, DE, USA). Either 50 ng (cell fractions) or 250 ng (PAXgene whole blood) of RNA was used for complementary DNA (cDNA) synthesis, which was performed using the RevertAid H Minus cDNA Synthesis Kit (Thermo Scientific, Waltham, MA, USA), according to the manufacturer’s protocol. Two CD19-enriched samples were excluded because of low RNA yield.