Computational protocol: Targeting DMPK with Antisense Oligonucleotide Improves Muscle Strength in Myotonic Dystrophy Type 1 Mice

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Protocol publication

[…] RNA was isolated from human fetal normal cells treated with both ASOs using QIAzol (QIAGEN). DNA microarray analyses were carried out with Affymetrix Human Gene 1.0 ST arrays (Affymetrix) according to their standard protocol. RNA from DMSXL mice treated with ISIS 486178 was isolated from tibialis anterior muscle using the proteinase K method. Six mice per condition were separated into two pooled groups for analysis. DNA microarray analyses were carried out with Affymetrix Mouse Gene 2.0 ST arrays according to the Affymetrix standard protocol. The amount of total RNA was measured using a NanoDrop ND-1000 spectrophotometer (Thermo Scientific). Optical density values at 260/280 were consistently above 1.9. Total RNA quality was assayed on an Agilent BioAnalyzer (Agilent Technologies). Briefly, 200 ng of total RNA per sample was labeled using the Affymetrix GeneChip WT cDNA Synthesis and Amplification Kit protocol and hybridized to the arrays as recommended by the manufacturer (Affymetrix). The cRNA hybridization cocktail was incubated overnight at 45°C while rotating in a hybridization oven. After 16-hr hybridization, the cocktail was removed and the arrays were washed and stained in an Affymetrix GeneChip fluidics station 450, according to the Affymetrix-recommended protocol. The arrays were scanned using the Affymetrix GCS 3000 7G and Gene-Chip Operating Software (Affymetrix) to produce the intensity files. The background subtraction and normalization of probe set intensities was performed using the method of robust multiarray analysis (RMA) described previously. For Affymetrix Human Gene 1.0 ST arrays, differentially expressed genes were identified by comparison of gene expression intensity using a moderated t test, and a Bayes smoothing approach was developed for a low number of replicates. To correct for the effect of multiple testing, the false discovery rate was estimated from p values derived from the moderated t test statistics. The analysis was performed using the affylmGUI Graphical User Interface for the Limma microarray package and GO analysis using the GO enrichment functionality in the Partek Genomics Suite software. For Affymetrix Mouse Gene 2.0 ST arrays, the CHP files were imported and analyzed with the Transcriptome Analysis Console Software (Affymetrix). Microarray analyses were performed by the CHU de Québec Research Center (Centre de Recherche du Centre Hospitalier Universitaire) Gene Expression Platform. […]

Pipeline specifications

Software tools affylmGUI, limma, Partek Genomics Suite, TAC Software
Application Gene expression microarray analysis
Organisms Mus musculus, Homo sapiens