Total RNA from Shp2hep−/− and WT mice liver was prepared with RNeasy mini kit (Qiagen Cat # 74104). Labeled cRNA was prepared from 500 ng RNA using the Illumina® RNA Amplification Kit from Ambion (Austin, TX, USA). The labeled cRNA (750 ng) was hybridized overnight at 58°C to the Sentrix Mouse -8 Expression BeadChip (>23,000 gene transcripts; Illumina, San Diego, CA, USA) according to the manufacturer’s instructions. BeadChips were subsequently washed and developed with fluorolink streptavidin-Cy3 (GE Healthcare). BeadChips were scanned with an Illumina BeadArray Reader. The microarray data have been deposited in the Gene Expression Omnibus (GEO) under the accession number of GSE51860. The gene expression data (GSE20599) for FXR−/−/SHP−/− DKO mice at 5 weeks of age were downloaded from the GEO (Anakk et al., 2011 (link)), processed with BeadStudio software and quantile normalized. The data (GSE29426) for FGF15/19-treated mice were downloaded from GEO (Potthoff et al., 2011 (link)) and processed with MAS5 algorithm (Affymetrix). Probes were filtered with detection p-value > 0.01 (for Shp2hep−/− and FXR−/−/SHP−/− DKO data) or with ABS call (for FGF15/19 data) before further analysis. Transcripts shared between datasets were used for K-means clustering with Cluster 3.0 software. Heat maps were generated with Java TreeView. GO analysis was performed with DAVID v.6.7 program.
Mas5 algorithm
Manufactured by Thermo Fisher Scientific
The MAS5 algorithm is a statistical method used for the analysis of microarray data. It is designed to provide an estimate of the expression level of a gene based on the measured probe intensities. The core function of the MAS5 algorithm is to perform background correction, normalization, and summarization of probe-level data to generate a single expression value for each gene.
Automatically generated - may contain errors
Lab products found in correlation
Genechip rice genome array, by Thermo Fisher Scientific (2 mentions)
Rneasy mini kit, by Qiagen (1 mentions)
Beadarray reader, by Illumina (1 mentions)
Fluorolink streptavidin cy3, by GE Healthcare (1 mentions)
Rna extraction reagent, by Qiagen (1 mentions)
Genechip scanner 3000, by Thermo Fisher Scientific (1 mentions)
Genechip operating software version 1, by Thermo Fisher Scientific (1 mentions)
6 protocols using mas5 algorithm
1
Transcriptional profiling of Shp2-deficient mouse liver
2
Transcriptional Profiling of Gastric Cancer
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Primary gastric tumors were obtained from National Cancer Centre, Singapore with approvals from Research Ethics Review Committee, and signed patient informed consent. Total RNA was extracted using Qiagen RNA extraction reagents (Qiagen, Venlo, Limburg, Netherlands) according to the instructions of the manufacturer and hybridized to Affymetrix Human Genome U133 plus Genechips (HG-U133 Plus 2.0, Affymetrix, Santa Clara, CA, USA). Raw data obtained after chip-scanning was pre-processed using the MAS5 algorithm (Affymetrix). Data were subjected to Log 10 transformation followed by median-centered across all probe sets for each sample (primary tumor). The centering is such that the median of expression in each sample is zero. 160 were classified by pathological diagnosis into diffuse-type GCs (68 cases) and intestinal-type (92 cases).
Formalin-fixed and paraffin-embedded (FFPE) primary tumor and matched normal tissue samples from 118 patients with gastric cancer were obtained from the Department of Pathology at the National University Hospital System, Singapore, under an institutionally approved protocol.
Formalin-fixed and paraffin-embedded (FFPE) primary tumor and matched normal tissue samples from 118 patients with gastric cancer were obtained from the Department of Pathology at the National University Hospital System, Singapore, under an institutionally approved protocol.
3
Transcriptome Analysis Using Affymetrix U133 Plus 2.0
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The transcriptome analyses used Human Genome U133 Plus version 2.0 high-density oligonucleotide arrays (Affymetrix, Santa Clara, CA, USA) with 54,000 probe sets and 1,300,000 distinct oligonucleotides to interrogate 47,000 well-characterized human transcripts. The sample labeling, microarray hybridization, and washing were performed on the basis of the standard protocols of the manufacturer. Briefly, total RNA was transcribed to double-strand cDNA and then to synthesized cRNA and labeled with cyanine-3-CTP. The labeled cRNAs were hybridized onto the microarray. After the slides were washed, the arrays were scanned by GeneChip Scanner 3000 (Affymetrix). The gene expression array data were digitalized by using GeneChip Operating Software (version 1.4; Affymetrix) and normalized by eliminating the highest and lowest 2% of the data by using MAS5 algorithm (Affymetrix). The microarray data have been deposited in the Gene Expression Omnibus (GEO) of the National Center for Biotechnology Information (NCBI) and are accessible through GEO series accession number GSE56649.
4
Transcriptome Analysis of Rice Embryo and Endosperm
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Total mRNAs were isolated from three replicates of 100 embryos and 50 endosperms and hybridizations on the Affymetrix GeneChip® Rice Genome Array (Affymetrix, Santa Clara, CA, USA) were performed as previously described (Galland et al., 2014a (link)). To obtain presence/absence calls for each probe, we normalized the CEL files by the MAS5 algorithm (Affymetrix). The CEL files were then normalized with the GC-RMA algorithm using the “gcrma” library available from the R Bioconductor suite of open-source softwares (Huber et al., 2015 (link)). To determine differentially expressed genes in the embryo and endosperm transcriptomes, we performed a usual two group t-test that assumes equal variance between groups. The raw P-values were adjusted by the Bonferroni method. We considered a gene as differentially expressed if adjusted-value is < 0.01. To establish the Pearson correlation, we plotted the embryo against the endosperm normalized mean probe intensity. All raw CEL files are available from the Gene Expression Omnibus under the accession GSE43780 (for the embryo: GSM1071216 , GSM1071217 , GSM1071204 ; for the endosperm: GSM1071199 , GSM1071201 , GSM1071210 ).
5
Comparative Transcriptomics of Rice Embryo and Endosperm
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Total mRNAs were isolated from three replicates of 100 embryos and 50 endosperms and hybridizations on the Affymetrix GeneChip® Rice Genome Array (Affymetrix, Santa Clara, CA, USA) were performed as previously described (Galland et al., 2014 (link), 2017 (link)). To obtain presence/absence calls for each probe, the CEL files were normalized by the MAS5 algorithm (Affymetrix). The CEL files were then normalized with the GC-RMA algorithm using the “gcrma” library available from the R Bioconductor suite of open-source software (Huber et al., 2015 (link)). Differentially expressed genes in the embryo and endosperm transcriptomes were detected by using a two-group t-test, followed by the Bonferroni method (adjusted P-value is < 0.01). All raw CEL files are available from the Gene Expression Omnibus under the accession GSE43780.
6
Gene Expression Analysis with GX12.6
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The gene expression data were analyzed with GX12.6 (Agilent Technologies). Raw data were summarized with the MAS5 algorithm (Affymetrix) and were normalized to log-transformed and median-centered data for the numerical analysis to permit gene selection. The differentially expressed probe sets used in the supervised hierarchical clustering were selected on the basis of P \ 0.05 and fold changes greater than 2.0. P values were calculated by a one-way ANOVA with Benjamini and Hochberg multiple correction. For the hierarchical clustering, average linkage clustering with the Pearson correlation distance was performed.
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