Computational protocol: Inhibition of Thrombopoietin/Mpl Signaling in Adult Hematopoiesis Identifies New Candidates for Hematopoietic Stem Cell Maintenance

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Protocol publication

[…] LSK cells were isolated from dnMpl and control transplanted mice by fluorescent activated cell sorting. For each sample, BM of 2–5 mice were pooled. RNA was isolated using RNeasy Micro Kit (Qiagen GmbH, Hilden, Germany). RNA quality was assessed using the Agilent 2100 Bioanalyzer. RNA was reverse transcribed, amplified and labelled using the Nugen Ovation Pico and Encore biotin kits (Nugen Technologies, AC Bemmel, Netherlands). The resulting material was hybridized to Affymetrix Mouse 430 2.0 arrays. Data were analyzed using R and Bioconductor []. Array quality was checked with the ArrayQualityMetrics package []. Arrays were background corrected, normalized and summarized using RMA []. We used LIMMA to detect differentially expressed probe sets applying the Benjamini-Hochberg procedure for correction of multiple testing. For testing enrichment of gene sets the Broad Institute GSEA software package was employed []. The datasets were collapsed into single genes and rank-ordered by signal to noise ratio. 1000 Gene set permutations were used to estimate statistical significance. Analyzed gene sets were obtained from MSigDB and GeneSigDB [,]. The comparison of gene expression profiles in dnMpl LSK cells in CD34+ cells of patients with severe aplastic anemia (SAA) and refractory cytopenia (RC) was made on the basis of the signal to noise ratio score given in the rank ordered gene lists derived with the GSEA software package. […]

Pipeline specifications

Software tools arrayQualityMetrics, limma, GSEA
Databases GeneSigDB MSigDB
Organisms Mus musculus, Homo sapiens
Diseases Anemia, Aplastic, Hematologic Diseases, Thrombocytopenia