Gc scanner 3000

The preparation of total RNA from hearts was performed hearts using the RNeasy Midi Kit (Qiagen, Hilden, Germany). The extraction procedure was conducted in accordance with the manufacturer’s manual. RNA integrity was checked with RNA Nano LabChips on an Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA). All samples in this study showed high quality RNA Integrity Numbers (RIN; mean 8.9). RNA was further analyzed by photometric Nanodrop measurement and quantified by fluorometric Qubit RNA assays (Life Technologies).
Synthesis of biotin labeled cDNA was conducted on three replicates of both experimental groups according to the manufacturers´ protocol (WT Plus Reagent Kit; Affymetrix, Inc). Briefly, 200 ng of total RNA were converted to cDNA. After amplification by in vitro transcription and another cDNS synthesis cycle, cDNA was fragmented and biotin labeled by terminal transferase. Finally, cDNA was hybridized to Affymetrix Mouse Transcriptome 1.0 arrays, washed and stained and scanned on the GC Scanner 3000 with G7 update as described in the standard Affymetrix GeneChip protocol (Version 2). Signal summarization using the RMA algorithm as well as statistical analysis were performed using the Affymetrix TAC software. The significance threshold was set to p < 0.05 and 0.01.

RNA was isolated using the RNeasy Mini Kit (Qiagen, Düsseldorf, Germany). Gene expression profiling (GEP) was performed using high-density oligonucleotide arrays (Clariom S mouse, Affymetrix, Santa Clara, CA, USA). Target preparation was performed according to the Affymetrix WTPlus Expression Protocol with approximately 100 ng of total RNA for the analysis on the Affymetrix Clariom S mouse array. Hybridization, washing, and staining of the arrays were done according to the Affymetrix recommendations on the GC Scanner 3000 with G7 update.
To identify differentially expressed genes, statistical analyses were performed in which the experimental samples were compared to the respective baseline samples by using the RMA signal summarization method and ANOVA test implemented in the Affymetrix Transcriptome Analysis Suite (Folder 3_TAC_analysis).
Gene set enrichment analysis (GSEA) was performed with RMA signals calculated with the Affymetrix Expression Console, GSEA software version 4.1, and MSigDB geneset collection v7.4 using default settings and gene set permutation. Pathways showing a false discovery rate (FDR) <0.25 and a normalized enrichment score (NES) > 1.5 were identified.