To disclose the molecular mechanisms underlying syndecan-1 nuclear translocation, we performed microarray analysis on cells that overexpressed FLs1 and NLSdel at similar levels. RNAs isolated from the cells were subjected to microarray analysis with the GeneChip Human Gene 1.1 ST Array (Affymetrix Inc., Santa Clara, CA, USA), which covered the whole transcript. Target synthesis and hybridization was performed in the Affymetrix core facility (Novum, Karolinska Institutet, Stockholm, Sweden). The raw data has been deposited in the MIAME compliant database Gene Expression Omnibus (accession number GSE81504). Image analysis and data pre-processing was performed with the Affymetrix Gene Chip Command Console. For data processing, we performed background correction with the PM-GCBG method (subtracting the GC-content specific background); data normalization with the Global Median method; and raw intensity value summarizations with PLIER (Probe Logarithmic Intensity Error). For each sample, the analysis generated a signal that represented the relative measure of transcript abundance. Individual signals that exceeded a value of 10 were considered for further analysis.
Genechip human gene 1.1 st array
Manufactured by Thermo Fisher Scientific
Sourced in United States, Netherlands
The GeneChip Human Gene 1.1 ST arrays are high-density oligonucleotide arrays designed for gene expression analysis. The arrays contain probes targeting over 19,000 well-annotated genes from the human genome. The arrays provide comprehensive coverage of the human transcriptome and enable the detection and quantification of gene expression levels.
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Lab products found in correlation
Rneasy micro kit, by Qiagen (2 mentions)
Trizol reagent, by Thermo Fisher Scientific (2 mentions)
Ambion wt expression kit, by Thermo Fisher Scientific (2 mentions)
Genechip command console, by Thermo Fisher Scientific (1 mentions)
Messageamp 2 biotin enhanced kit, by Thermo Fisher Scientific (1 mentions)
Whole transcript cdna synthesis, by Thermo Fisher Scientific (1 mentions)
Nanodrop nd 1000 spectrophotometer, by Agilent Technologies (1 mentions)
Agilent 2100 bioanalyser, by Agilent Technologies (1 mentions)
Agilent 2100 bioanalyzer, by Agilent Technologies (1 mentions)
Affymetrix genechip wt terminal labeling kit, by Thermo Fisher Scientific (1 mentions)
Bioanalyzer 2100 device, by Agilent Technologies (1 mentions)
Genetitan instrument, by Thermo Fisher Scientific (1 mentions)
6 protocols using genechip human gene 1.1 st array
1
Microarray Analysis of Syndecan-1 Translocation
2
Transcriptomic profiling of lean and obese PBMC
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PBMC samples from 15 lean and 29 abdominally obese subjects yielded enough RNA of sufficient quality at all collection points to perform microarray analysis. Microarray analysis was performed for each individual at fasting (T0) and 4 h (T4) in the postprandial state. The 4 h timepoint was chosen for transcriptomics analysis to allow for comparison to previous studies (Esser et al. 2015 (link)). Total RNA was labelled using a one-cycle cDNA labelling kit (MessageAmp™ II-Biotin Enhanced Kit; Ambion, Inc., Nieuwerkerk a/d IJssel, Netherlands) and hybridized to GeneChip® Human Gene 1.1 ST Array targeting 19 738 unique gene identifiers (Affymetrix, Inc., Santa Clara, CA, USA). Sample labelling, hybridization to chips, and image scanning were performed according to the manufacturers’ instructions.
3
Affymetrix GeneChip Transcriptome Analysis
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Hybridization to Affymetrix GeneChip Human Gene 1.1 ST Arrays, washing, and scanning were performed according to the manufacturer (Affymetrix, Santa Clara, CA). Data analysis was performed on CEL files using Partek® Genomics Suite TM (Partek Inc., St. Louis, MO). Data were normalized with the multi-average method45 (link). Batch effect removal was applied for the different samples, followed by one-way ANOVA. Clustering analysis was performed by Partek Genomics Suite software with Pearson’s dissimilarity correlation by average linkage methods. Functional annotation was performed using the DAVID software. The raw data was deposited in the GEO database (accession number GSE69658).
4
RNA Isolation and Microarray Analysis
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RNA of the mucosal biopsies was isolated using TRIzol reagent (Life technologies, Bleiswijk, Netherlands) and further purified using the RNeasy micro kit (Qiagen, Venlo, Netherlands). RNA yield was measured with the Nanodrop ND-1000 Spectrophotometer, and the quality of the RNA samples was verified with an Agilent 2100 Bio analyser (Agilent Technologies, Amstelveen, Netherlands). One hundred nanogram of RNA was used for whole transcript cDNA synthesis (Affymetrix, Inc., Santa Clara, USA). Hybridization, washing and scanning of Affymetrix GeneChip Human Gene 1.1 ST arrays was carried out according to standard Affymetrix protocols.
5
Transcriptomic Analysis of Skeletal Muscle
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Total RNA was isolated from the skeletal muscle tissue by using Trizol reagent (Invitrogen, Breda, Netherlands). Thereafter, RNA was purified using the Qiagen RNeasy Micro kit (Qiagen, Venlo, Netherlands), and RNA quality was checked using an Agilent 2100 bioanalyzer (Agilent Technologies, Amsterdam, Netherlands). Total RNA (100 ng) was labelled using an Ambion WT expression kit (Life Technologies, Bleiswijk, Netherlands) and hybridized to human whole genome Genechip Human Gene 1.1 ST arrays coding 19.732 genes, (Affymetrix, Santa Clara, CA). Sample labelling, hybridization to chips, and image scanning were performed according manufacturer's instructions.
6
Robust RNA Quality Assessment for Microarray
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RNA quantity and quality was assessed spectrophotometrically (Nanodrop) and with 6000 Nano chips via a Bioanalyzer 2100 device (Agilent, Santa Clara, CA, USA), respectively. RNA was judged as being suitable for array hybridization only if samples showed intact bands corresponding to the 18S and 28S ribosomal RNA subunits, displayed no chromosomal peaks or RNA degradation products, and had a RIN (RNA integrity number) above 8.0. The Ambion WT Expression kit (Life Technologies, cat. no. 4411974) in conjunction with the Affymetrix GeneChip WT Terminal Labeling kit (Affymetrix, Santa Clara, CA; cat. no. 900671) was used for the preparation of labeled cDNA from 100 ng of total RNA without rRNA reduction. Labeled samples were hybridized on Affymetrix GeneChip Human Gene 1.1 ST arrays that contain 30,000 coding transcripts and over 11,000 long intergenic non-coding transcripts, provided in plate format. Hybridization, washing, and scanning of the array plates was performed on an Affymetrix GeneTitan Instrument, according to the manufacturer's recommendations. Quality control of the hybridizations to the Human Gene 1.1 ST array and primary data analysis were performed according to strict criteria to ensure that the array data were of the highest possible quality.
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