Computational protocol: Transcriptional regulation of gene expression during osmotic stress responses by the mammalian target of rapamycin

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Protocol publication

[…] NIH3T3-immortalized wild-type and NFAT5-deficient MEFs () from passages 30–35 were plated (175 000 cells in 60-mm dishes) in isotonic medium and 2 days later were either left untreated or treated for 8 h with 500 mOsm/kg. When indicated, the mTOR catalytic inhibitor Torin1 was added 1 h before hypertonicity (500 mOsm/kg) treatment. Cells were lysed in RLT buffer (300 µl, RNeasy system, QIAGEN Cat. 74 104) and total RNA was isolated using the manufacturer protocol. The RNA integrity was assessed using Agilent 2100 Bioanalyzer (Agilent), and only samples with high integrity [RNA integrity number (RIN) > 7.5] were subsequently used in microarray experiments. Amplification, labeling and hybridizations were performed according to protocols from Ambion and Affymetrix. Briefly, 250 ng of total RNA were amplified using the Ambion® WT Expression Kit (Ambion/Applied Biosystems), labeled using the WT Terminal Labeling Kit (Affymetrix Inc), and then hybridized to Mouse Gene 1.0 ST Array (Affymetrix) in a GeneChip® Hybridization Oven 640. Washing and scanning were performed using the Hybridization Wash and Stain Kit and the GeneChip® System of Affymetrix (GeneChip® Fluidics Station 450 and GeneChip® Scanner 3000 7G). Three independent microarray hybridizations were performed for each experimental condition: cells maintained at 300 mOsm/kg or exposed to 500 mOsm/kg (8 h) without or with 100 nM Torin1 in the case of wild-type MEFs, or 300 mOsm/kg and 500 mOsm/kg for Nfat5−/− MEFs. Microarray data analysis was performed as follows: after quality control of raw data, it was background-corrected, quantile-normalized and summarized to a gene level using the robust multi-chip average (RMA) () obtaining a total of 28 853 transcript clusters, excluding controls, which roughly correspond to genes. Core annotations (version NetAffx 30, human genome 18) were used to summarize data into transcript clusters. Linear Models for Microarray (LIMMA) (), a moderated t-statistics model, was used for detecting differentially expressed genes between the conditions in study. Correction for multiple comparisons was performed using false discovery rate. Genes with an adjusted P-value < 0.05 or with a P < 0.01 for those comparisons with few results after adjusting P-values were selected as significant. Hierarchical cluster analysis was also performed to analyze how data aggregated and linear model for regression purposes. All data analysis was performed in R (version 2.11.1) with packages aroma.affymetrix, Biobase, Affymetrix, LIMMA and genefilter. The microarray data have been deposited in NCBI's Gene Expression Omnibus and are accessible through GEO Series accession number GSE27485. […]

Pipeline specifications

Software tools limma, Aroma.affymetrix, Biobase, genefilter
Databases GEO
Application Miscellaneous
Organisms Homo sapiens
Chemicals Sirolimus