Abstract
This protocol details a method to analyze two tissue samples at the transcriptomic level using microarray analysis, ingenuity pathway analysis (IPA) and gene set enrichment analysis (GSEA). Methods such as these provide insight into the mechanisms underlying biological differences across two samples and thus can be applied to interrogate a variety of processes across different tissue samples, conditions, and the like. The full method detailed below can be applied to determine the effects of muscle-specific Notch1 activation in the mdx mouse model and to analyze previously published microarray data of human liposarcoma cell lines.
Highlights
[Background] Transcriptomic analysis of various cell types is crucial to elucidate the functional elements of a cell, provides insight into cell-specific characteristics and can highlight changes associated with different development or disease stages (Wang et al, 2009)
The methods described below were used by Bi and colleagues to understand the effects of Notch signaling in muscle regeneration and liposarcoma, a common soft-tissue cancer type (Bi et al, 2016a)
Bi and colleagues performed microarray analysis, ingenuity pathway analysis (IPA) and gene set enrichment analysis (GSEA) to find that over-activation of Notch in mouse inguinal white adipose tissue shares signatures of human liposarcoma (Bi et al, 2016b)
Summary
[Background] Transcriptomic analysis of various cell types is crucial to elucidate the functional elements of a cell, provides insight into cell-specific characteristics and can highlight changes associated with different development or disease stages (Wang et al, 2009). Note: Primers used to validate microarray results and for real-time quantitative PCR in Bi et al (2016b) are listed in Supplemental file. Add 4.7 μl cDNA reaction to each tube containing RNA + Spike Mix (final volume is 10 μl), mix by pipetting and briefly spin down.
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