Rna 6000 nano lab on a chip kit

Total RNA was isolated using a mirVanamiRNA Isolation Kit (Ambion, Austin, TX) according to the manufacturer's directions. The purity and concentration of RNA were determined from OD260/280 readings byGenequant Pro Classic spectrophotometer (GE Healthcare, Little Chalfont, UK). RNA integrity was determined by capillary electrophoresis using the RNA 6000 Nano Lab-on-a-Chip kit and the Bioanalyzer 2100 (Agilent Technologies, Santa Clara, CA). Only RNA extracts with RNA integrity no. values ≥6 underwent further analysis.

Nuclear acid extraction was performed as described previously in detail [13 (link)]. Briefly, total RNA was extracted from individual hearts (n = 5-6 hearts per experimental group, 16 samples in total, with normalized glucose levels at <13mM) using glass beads and RNAzol (Campro Scientific, Veenendaal, The Netherlands). RNA integrity was examined using the RNA 6000 Nano Lab-on-a-Chip kit and a bioanalyzer 2100 (Agilent Technologies, Amstelveen, The Netherlands). The Illumina^® TotalPrep™ RNA Amplification Kit (Ambion, art.No.AM-IL1791) was used to synthesize biotin labeled cRNA starting with 500 ng total RNA. The concentration of the labeled cRNA was measured using the Nanodrop spectrophotometer. The amount of biotinylated cRNA which was hybridized onto the MouseRef-8 Expression BeadChip was 750 ng. Illumina’s Genomestudio v1.1.1 software with the default settings advised by Illumina was used for Gene Expression analysis. All the quality control data of this BeadChip were within specifications of the microarray service provider (Service XS, Leiden, the Netherlands).

Total RNA was extracted from adipose tissues using glass beads and RNAbee (Tel-Test Inc, Friendswood, USA). RNA integrity was examined using the RNA 6000 Nano Lab-on-a-Chip kit and a Bioanalyzer 2100 (Agilent Technologies^®, Amstelveen, The Netherlands). The Illumina^® TotalPrep™ RNA Amplification Kit (Ambion, art.No.AM-IL1791) was used to synthesize biotin labeled cRNA starting with 500 ng total RNA. The biotinylated cRNA was then hybridized onto the MouseRef-8 Expression BeadChip was 750 ng. Lastly, the default setting of Illumina’s Genomestudio v1.1.1 was used for Gene Expression analysis. All the quality control data of this BeadChip were within specifications of the microarray service provider (Service XS, Leiden, the Netherlands). The microarray gene expression data were validated using quantitative real-time PCR for Leptin using established protocols and primer/probe sets45 (link). Ingenuity Pathway Analysis (IPA) was used to analyse microarray data and Pparg2 target genes. The upstream regulator analysis tool of IPA was used to determine the transcriptional activity of Pparg2 transcription factor essentially as reported45 (link). A negative Z-score <−2 indicated a reduced transcriptional activity based on the direction of gene expression changes of target genes.