Rneasy total rna mini kit

The material for the study consisted of total RNA extracted from the cell cultures with the TRIzol reagent (Invitrogen). The isolated RNA was purified with the RNeasy Total RNA Mini Kit and digested with DNase I (Qiagen) in order to eliminate any possible contamination with DNA. Approximately 8 μg of total RNA was used for the double-stranded cDNA synthesis. Biotin-labeled cRNA was synthesized and biotinylated cRNA was then fragmented and hybridized with the Test3 microarray and HG U133A (Affymetrix) and labeled twice with the streptavidin-phycoerythrin conjugate and biotinylated streptavidin antibodies.
The fluorescence intensity was analyzed with the GeneArray Scanner G2500A. The quantity and quality of total RNA, cDNA, and cRNA were assessed spectrophotometrically and by means of the electrophoresis technique in 1% agarose gel.

Total RNA was prepared from cell lysates using the RNeasy total RNA Mini kit (Qiagen, Courtaboeuf, France), according to the manufacturer’s protocol. The amount and purity of total RNA was determined (NanoDrop, Thermo Fisher Scientific, Waltham, MA, USA) and the integrity of the RNA was assessed using a 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA). One-Colour Microarray-Based Gene Expression Analysis Protocol (Agilent Technologies) was used to amplify and label RNA. Samples were hybridised to whole human genome microarray 4 x 44K (G4112F, Agilent Technologies). For each condition (one control (DMSO) and two treatments (ETP-45658 and PI-103), four replicate hybridisations were carried out. Arrays were scanned at 5 μm resolution on an Agilent DNA Microarray Scanner (G2565BA, Agilent Technologies). Microarray data has been deposited at Gene Expression Omnibus (GEO) (accession number GSE56579).

Subcutaneous adipose samples were collected at baseline and week 6 by needle biopsy from the periumbilical region under local anesthesia. Samples were frozen rapidly in liquid nitrogen then stored at -80°C until they were further processed for microarray analysis as described in Rizkalla et al. (2012) (link). Briefly, total RNA was extracted by using the RNeasy total RNA Mini kit (QIAGEN) with one-column DNase digestion. RNA quality and concentration were assessed by using an Agilent 2100 Bioanalyzer (Agilent Technologies). An Illumina RNA amplification kit (Ambion) was used according to the manufacturer’s instructions to obtain biotin-labeled complementary RNA from 250 ng total RNA. Hybridization processes were performed with Illumina Human HT-12 version 3.0 Expression BeadChips (Illumina, Inc.). Hybridized probes were detected with cyanin-3-streptavidin (1 mg/mL; Amersham Biosciences, GE Health Care) and scanned by using an Illumina BeadArray Reader. Raw data were extracted with GenomeStudio 2011.1 Software by using the default settings and quantile normalization. Relevant genes associated with changes in IS were annotated using the FunNet tool (Prifti et al., 2008 (link)), using the whole human genome as the reference gene set.