Geneatlas microarray system

After euthanasia, mouse ventricular tissue from 3 to 4-monthold mice was harvested, and then, stored at −80°C. When all samples had been collected, total RNA was collected, reverse-transcribed into cDNA, and then, analyzed by “wholetranscript transcriptomics” using the GeneAtlas microarray system (Affymetrix, Santa Clara, CA, USA) and manufacturer’s protocols. Mogene 1.1 ST array strips (Affymetrix) were used for hybridization with newly synthesized sscDNA. Each microarray comprised 770 317 distinct 25-mer probes to probe an estimated 28 853 transcripts, with a median 27 probes per gene. Gene expression changes that were associated with cardiac-specific Kcne2 deletion were analyzed using Ingenuity Pathway Analysis (Qiagen, Hilden, Germany) to identify biological networks, pathways, processes, and diseases that were most highly represented in the differentially expressed gene (DEGs) identified. Expression changes of ≥1.5-fold and P < .05 were included in the analysis.

We performed microarray experiments and analysis as previously described.^{35 (link)} Whole brain tissues were dissected from male mice. Total RNA was extracted (Qiagen, Germantown, MD, USA) according to the manufacturer’s protocol. RNA samples with A260/A280 absorbance ratios between 2.00 and 2.20 were reverse-transcribed into cDNA and analyzed by ‘whole-transcript transcriptomics’ using the GeneAtlas microarray system (Affymetrix, Santa Clara, CA, USA) and the manufacturer’s protocols.
MoGene 1.1 ST array strips (Affymetrix) were used to hybridize to newly synthesized sscDNA.
Each array comprised 770 317 distinct 25-mer probes to probe an estimated 28 853 transcripts, with a median 27 probes per gene.
Gene expression changes associated with the methionine treatment were analyzed with transcriptome analysis console software (Affymetrix) using Tikey’s Bi-weight Average algorithm and default settings, generating fold-change and unpaired ANOVA values, reported in Supplementary Tables S1.1 and S1.2. Gene expression changes did not reach P<0.05 after Benjamini-Hochberg step-up false discovery rate procedure to correct for multiple comparisons, given the relatively small number of samples and large number of genes tested. However, gene expression changes uncovered using the initial microarray screen were subsequently validated using real-time qPCR in the same samples (see Figures 4c–g).