Cyanin 3 streptavidin

The RNA preparation, cRNA generation and microarray hybridization procedures were used as previously described [24 (link)]. In brief, Trizol-reagent (Invitrogen, Carlsbad, CA, USA) was used to isolate total RNA from the twin muscle biopsies, which were homogenized on FastPrep FP120 apparatus (MP Biomedicals, Illkirch, France).
An Illumina RNA amplification kit (Ambion, Austin, TX, USA) was used according to the manufacturer’s instructions to obtain biotinlabeled cRNA from 500 ng of total RNA.
Hybridizations to Illumina HumanWG-6 v3.0 Expression BeadChips (Illumina Inc., San Diego, CA, USA) containing probes for PLIN2 and PLIN5, were performed by the Finnish DNA Microarray Center at Turku Center for Biotechnology according to the Illumina BeadStation 500x manual.
Hybridized probes were detected with Cyanin-3-streptavidin ( $1\mathsf{\mu }\mathrm{g}·{\mathrm{mL}}^{-1}$ , Amersham Biosciences, GE Healthcare, Uppsala, Sweden) using Illumina BeadArray Reader (Illumina Inc.) and BeadStudio v3 software (Illumina Inc.).
The gene expression data and the raw data sets for skeletal muscle have been deposited in the GEO database (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE20319, accessed on 5 January 2023).

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.

RNA concentration and integrity were assessed with the Agilent 2100 Bioanalyzer (Agilent Technologies) and amplified with the Illumina RNA amplification kit according to the manufacturer's protocol (Ambion) to obtain biotin-labeled complementary RNA from 250 ng total RNA. Hybridization processes were performed with Illumina MouseRef-8 v2.0 Expression BeadChip (Illumina Inc). Hybridized probes were detected with cyanin-3-streptavidin (1 mg/mL; Amersham Biosciences, GE Health Care) and scanned using an Illumina BeadArray Reader. Raw data were extracted with GenomeStudio 2011.1 software using the default settings and without any additional normalization.
The difference in gene expression between the two conditions (NO or IH) was determined using SAM software (Significant Analysis of Microarray, Stanford University, CA, USA, https://statweb.stanford.edu/~tibs/SAM/), which provides a list of significant genes and an estimate of the false discovery rate (FDR) representing the percentage of genes that could be identified by chance. The FDR was tested at 5%.