Following euthanization by inhalation of CO2 and cervical dislocation, lumbar DRGs (L1–L6) were isolated from five mice bearing four genomic copies of the mutant Zfhx2 gene and from seven wild-type controls. Total RNA was isolated using the PureLink™ RNA Micro Kit (Invitrogen) and run on the GeneChip Mouse Transcriptome Array 1.0 (Affymetrix). Expression data were analysed using the Expression and Transcriptome Analysis Consoles (Affymetrix). Microarray data have been deposited at Gene Expression Omnibus Array Express for public use with reference number E-MTAB-5650.
Expression and transcriptome analysis console
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Expression and Transcriptome Analysis Consoles are lab equipment designed for the analysis of gene expression and transcriptome data. They provide a platform for researchers to perform various tasks related to the study of gene expression patterns and transcriptome-level analysis.
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Lab products found in correlation
Exon 2.0 st array, by Thermo Fisher Scientific (2 mentions)
Lightcycler 480, by Roche (2 mentions)
Genechip mouse transcriptome array 1, by Thermo Fisher Scientific (1 mentions)
Purelink rna micro kit, by Thermo Fisher Scientific (1 mentions)
Ezinfo, by Sartorius (1 mentions)
Mse data viewer, by Waters Corporation (1 mentions)
Progenesis qi, by Waters Corporation (1 mentions)
Progenesis qi, by Sartorius (1 mentions)
Masslynx, by Waters Corporation (1 mentions)
Sigmaplot, by Grafiti LLC (1 mentions)
Sigmastat, by Grafiti LLC (1 mentions)
Genechip wt pico reagent kit, by Thermo Fisher Scientific (1 mentions)
Genechip human transcriptome array 2.0 hta, by Thermo Fisher Scientific (1 mentions)
Genechip scanner 3000, by Thermo Fisher Scientific (1 mentions)
Goscript reverse transcriptase kit, by Promega (1 mentions)
Rneasy mini kit, by Qiagen (1 mentions)
Nucleospin rna mini kit, by Macherey-Nagel (1 mentions)
Goscript reverse transcriptase, by Promega (1 mentions)
Maxima reverse transcriptase, by Thermo Fisher Scientific (1 mentions)
Sybr green, by Promega (1 mentions)
6 protocols using expression and transcriptome analysis console
1
Transcriptome Analysis of Mutant Zfhx2 Mice
2
Transcriptomic and Lipidomic Analysis of Human Meibum
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The transcriptomic datasets were processed using Expression and Transcriptome Analysis Consoles (v.4.0.1.36; both from Affymetrix) and SigmaStat (v.3.5, from Systat Software, Inc., San Jose, CA, USA). The default (and currently the industry standard) filter criteria: (1) (+2) < LFC < (−2), and (2) ANOVA p-value (condition pair) ≤0.05, were used to analyze the data. A tighter LFC of >(+1.2) and <(−1.2), as proposed in [34 (link)], was also tested, but deemed impractical because of an unrealistically high number of samples needed to satisfy statistical criteria (see Discussion).
The RP-UPLC/MS data were analyzed using MassLynx (v.4.1), MSe Data Viewer (v.1.4), and Progenesis QI software packages (from Waters). ASupplemental Table S1 lists major lipids of human meibum relevant to this study, and their corresponding m/z values. SigmaStat and SigmaPlot software packages from Systat Software, Inc. were used to conduct statistical evaluation of the data.
The transcriptomic and lipidomic data for two genders were compared gender-wide using Student’s t-test for the two groups. Tests with p-values ≤ 0.05 were considered statistically significant. Principal component analyses were performed using Transcriptome Analysis Console, Progenesis QI, and EZInfo (v.3.0.3.0 from Umetrics AB, Umeå, Sweden).
The RP-UPLC/MS data were analyzed using MassLynx (v.4.1), MSe Data Viewer (v.1.4), and Progenesis QI software packages (from Waters). A
The transcriptomic and lipidomic data for two genders were compared gender-wide using Student’s t-test for the two groups. Tests with p-values ≤ 0.05 were considered statistically significant. Principal component analyses were performed using Transcriptome Analysis Console, Progenesis QI, and EZInfo (v.3.0.3.0 from Umetrics AB, Umeå, Sweden).
3
Comprehensive Transcriptome Analysis using GeneChip
4
Quantitative Real-Time PCR Analysis
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Total RNA was prepared by the RNeasy Mini Kit from Qiagen or from Macherey-Nagel (Düren, Germany). cDNA synthesis and hybridization to Affymetrix Exon 2.0 ST array was performed by the group of Prof. Nürnberg (CCG, Cologne, Germany). The raw data were processed with the help of the Affymetrix Expression and Transcriptome analysis console. For quantitative real time PCR, 2 μg of RNA was reverse transcribed using random primer and Go-Script Reverse Transcriptase Kit (Promega). Real time PCRs were performed with SYBR Green and a Roche LightCycler 480 (Roche Diagnostics). The expression of the various factors was normalized against the housekeeping gene hypoxanthine guanine phosphoribosyltransferase (HPRT). The values obtained for cells transfected with control siRNA and grown under normoxia were set as 1 and the fold differences were calculated according to the comparative threshold method [27 (link)]. The primers that were used for RT-PCR are summarized in Table 1 .
5
Microarray Data Analysis Pipeline
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Microarray data analyses were carried out using Expression and Transcriptome Analysis Console (Affymetrix). Data normalization based on median signal intensities was qualified and quantified using Robust Multichip Analysis algorithm, which includes background adjustment, quantile normalization, and summarization. A log2 fold change >2 was considered as upregulated genes and a log2 fold change <−2 as downregulated genes. Statistical significance was calculated by one-way ANOVA and false discovery rate (FDR) correction (adjust p-value based on Benjamin–Hochberg Step-Up FDR-controlling Procedure). A p < 0.01 was considered statistically significant.
6
Quantitative RNA Expression Analysis
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Total RNA was isolated by the NucleoSpin® RNA Mini Kit from Macherey-Nagel (Düren, Germany). C-DNA synthesis and hybridization to Affymetrix Exon 2.0 ST array was performed by the group of Professor Nürnberg (CCG, Cologne, Germany). The raw data were processed with the help of the Affymetrix Expression and Transcriptome analysis console. For quantitative RT-PCR, 2 μg of RNA was reverse transcribed using random primer and the Go-Script Reverse Transcriptase (Promega) or the Maxima reverse transcriptase (Invitrogen). QRT-PCR was performed with the Go-Taq qPCR mastermix including Sybr green (Promega) and a Roche Light Cycler 480 (Roche Diagnostics). The sequences of the primers used for PCR are provided in Table 1 . The expression of the various factors was normalized against the house keeping gene hypoxanthine guanine phosphoribosyltransferase (HPRT). The fold changes of the relative gene expression values for the various factors in relation to cells transfected with siControl were calculated by Δ-comparative threshold method [40 (link)].
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