Total RNA was extracted using TRIzol (Thermofisher, Waltham, USA) and the Direct-zol RNA Miniprep kit (Zymo Research, Irvine, USA) according to the manufacturer’s instructions. Genome-wide expression analyses was performed using Clariom S arrays (Thermo Fisher Scientific) using 100 ng RNA of each sample according to the manufacturer’s recommendations (WT Plus Kit, Thermo Fisher Scientific). Data were analyzed using Transcriptome Analyses Console (Thermo Fisher Scientific, version 4.0) and expressed in log2 values. Sample MMI_ZT06_a and MMI_ZT18_d were removed from the data due to low quality.
Wtplus kit
Manufactured by Thermo Fisher Scientific
Sourced in United States
The WTPLus kit is a laboratory instrument designed for analytical testing. It provides core functionality for sample preparation and analysis. The WTPLus kit is intended for use in research and testing applications.
Automatically generated - may contain errors
Lab products found in correlation
Trizol, by Thermo Fisher Scientific (4 mentions)
Clariom s array, by Thermo Fisher Scientific (2 mentions)
Transcriptome analyses console, by Thermo Fisher Scientific (2 mentions)
Direct zol rna miniprep kit, by Zymo Research (2 mentions)
Expression console software, by Thermo Fisher Scientific (2 mentions)
Genechip fluidics station 450, by Thermo Fisher Scientific (2 mentions)
Genechip scanner 3000, by Thermo Fisher Scientific (2 mentions)
Transcriptome analysis console software, by Thermo Fisher Scientific (1 mentions)
Rneasy mini kit, by Qiagen (1 mentions)
Genechip human transcriptome array 2, by Thermo Fisher Scientific (1 mentions)
Transcriptome analysis console 4, by Thermo Fisher Scientific (1 mentions)
Genechip scanner 3000 7g, by Thermo Fisher Scientific (1 mentions)
Mouse gene 2.0 st array, by Thermo Fisher Scientific (1 mentions)
Rnase free dnase, by Thermo Fisher Scientific (1 mentions)
Rneasy micro kit, by Qiagen (1 mentions)
Genechip human clariom s array, by Thermo Fisher Scientific (1 mentions)
Clariom s mouse array, by Thermo Fisher Scientific (1 mentions)
Genespring 14, by Agilent Technologies (1 mentions)
Humanht 12 v4 beadchip, by Illumina (1 mentions)
Human clariom s assay, by Thermo Fisher Scientific (1 mentions)
12 protocols using wtplus kit
1
Transcriptome Analysis of RNA Samples
2
Microarray Analysis of Mouse Gastric Tissue
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Gastric corpus tissue from wild-type C57BL/6 littermate/cage mate mice was directly placed in RLT buffer containing 2-mercaptoethanol, and RNA was isolated using the RNeasy Mini Kit (QIAGEN), per the manufacturer’s instructions. Gene expression profiling was performed using microarray analysis in collaboration with the Genome Technology Access Core at the Washington University in St. Louis School of Medicine. RNA was amplified using the WT Plus kit (Thermo Fisher Scientific) and hybridized to Agilent 8 × 60 gene chips. All data were analyzed using the Transcriptome Analysis Console software (Thermo Fisher Scientific). Microarray data were uploaded to the National Center for Biotechnology Information’s Gene Expression Omnibus database with the following accession numbers: GSE190508, GSE190509, and GSE190563.
3
RNA Extraction and Microarray Analysis
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Total RNA was extracted using TRIzol (Thermo Fisher, Waltham, USA) and the Direct-zol RNA Miniprep kit (Zymo Research, Irvine, USA) according to the manufacturer’s instructions. Genome-wide expression analyses were performed using Clariom S arrays (Thermo Fisher Scientific) using 100 ng RNA of each sample according to the manufacturer’s recommendations (WT Plus Kit, Thermo Fisher Scientific). Data was analyzed using Transcriptome Analyses Console (Thermo Fisher Scientific, version 4.0) and expressed in log2 values.
4
Transcriptome Analysis using HTA 2.0
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After RNA isolation and QC, samples were labeled for the GeneChip Human Transcriptome Array 2.0 (HTA 2.0, Affymetrix). Labeling was performed with Affymetrix Proprietary DNA Label (biotin-linked) using a WT Plus Kit (Affymetrix) provided with the HTA 2.0, following the standard operating protocol for HTA 2.0, including PolyA controls. Hybridization, washing, and staining were performed with the WT Plus Kit, following the standard operating protocol for HTA 2.0. Washing and staining were performed using a GeneChip Fluidics 450. Scanning was performed with a GeneChip Scanner 3000. These data were deposited in the Gene Expression Omnibus under accession GSE85033.
5
Clariom S Mouse Array Transcriptome Analysis
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Mouse RNA samples were labeled using the Affymetrix WTPLus kit according to manufacturer’s guidelines, and probed using the Clariom S Mouse Array. Raw data generated from Clariom S Mouse Arrays were processed using Affymetrix Expression Console Software. CEL files containing feature intensity values were converted into summarized expression values using by Robust Multi-array Average (RMA) which consists of background adjustment, quantile normalization and summarization across all chips. All samples passed QC thresholds for hybridization, labeling and the expression of housekeeping gene controls. The complete normalized data set is available on GEO under Series accession number GSE103022.
6
Transcriptome Analysis of CAR T Cells
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Human peripheral blood T cells were grafted with the respective CAR and cultivated for 7 days. CAR T cells were flow sorted and stimulated through the CAR for 48 h by incubation in microtest-plates coated with the anti-idiotypic mAb BW2064 directed against the scFv of the CAR. RNA from 5 × 106 cells each was extracted, labeled using the Affymetrix WTPLus kit according to manufacturer's guidelines, and probed to the Clariom S Human Array. After staining, arrays were scanned using the Affymetrix Gene-Chip Scanner-3000-7G; quality control analysis was performed using Affymetrix GCOS software. Raw array data were processed using the Affymetrix Transcriptome Analysis Console (TAC) 4.0 software. CEL files containing feature intensity values were converted into summarized expression values using robust multi-array average (RMA), which consists of background adjustment, quantile normalization, and summarization across all chips. Expression data [log(2)] of CAR T cells were generated and x-fold expression of differentially expressed genes determined. Mean values of expression data [log(2)] of CAR T cells from three different donors were determined. Mean values with differential expression of ≥2.5 in mean and ≥2 in individual samples were regarded significant. Statistical analysis was performed by ANOVA and values with p < 0.05 were included in the analysis.
7
Insulin and IGF-1 Stimulation Microarray Analysis
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Microarrays were processed using the WT PLUS kit and Mouse Gene 2.0 ST arrays from Affymetrix. Total RNA input was 250 μg. For the final step, 5.5 μg cDNA were fragmented and labelled, and the chips were hybridized for 16 h in the GeneChip Hybridization Oven 645 using 3.5 μg of this fragmented and labelled product. Chips were then scanned using the GeneChip Scanner 7,000, and normalized using RMA and Affymetrix's AGGC software. Then bioinformatic analysis was done in R/Bioconductor47 examining paired differences between stimulated versus unstimulated samples. One pair was an extreme outlier in the principal component analysis plot and received a low quality weight48 , and thus was removed. However, all the cell lines were included for the further qPCR confirmation. Fold change of the expression of each probeset was calculated by comparing the gene expression after 6 h insulin or IGF-1 stimulation with the 6 h mock treated cells. Statistical significance of probe sets was assessed with empirical Bayesian linear modelling using the limma package49 , and significance of gene sets was assessed with the sigPathway package50 (link). Heatmaps were created with the gplots package, and volcano plots and scatterplots were created with the ggplot2 package51 .
8
Comparative Transcriptome Analysis of Zebrafish Embryos
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Total RNA was isolated from three biologically independent pools of WT and marsanne embryos using Trizol/chloroform extraction and isopropanol precipitation and treated with RNase-free DNase (Ambion). Any remaining phenol was removed using an RNeasy micro kit (Qiagen). Samples were provided to the Ramaciotti Centre for Genomics (University of New South Wales, Australia) for QC and processing. Input was 100 ng of total RNA, samples were processed using the Affymetrix WT Plus kit with no amplification and hybridization was to Affymetrix Zebrafish Gene Array 1.0 ST (Affymetrix). Data were analysed using Bioconductor (Bioconductor—Open Source Software for Bioinformatics ( http://www.bioconductor.org ) Copyright 2017) and R version 3.2.5 (The R Project for Statistical Computing ( https://www.r-project.org/ )) packages. Expression values for all genes were calculated using the robust multi-array average method57 (link). Data for biological replicates clustered into their separate groups corresponding to WT and marsanne. For the identification of genes with differential expression between groups, fold-change cutoff (≥2.0) and P value cutoff (≤0.05) were used for differential expression.
9
Transcriptome Analysis Using Affymetrix Microarray
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Total RNA (100 ng) was amplified and converted into biotinylated sense-strand cDNA targets using the Affymetrix WT PLUS kit according to the manufacturer’s instructions (Affymetrix, Santa Clara, CA, USA). All samples were collected using the same study protocol, irrespective of clinical features. We adhered to the manufacturer’s recommendations regarding RNA quality control (260/280 ratio between 1.7 and 2.1). The Nanodrop ND-1000 was used to monitor and normalise cDNA concentration across samples throughout the target preparation. Samples of different response status and time-point were arranged in the 96-well plate at random to avoid cDNA conversion bias. For each sample, 5-μg of fragmented, end-labelled sense-strand target cDNA was hybridised to a GeneChip Human Transcriptome Array (HTA) 2.0 before incubation for 16 h at 45 °C in the GeneChip® Hybridization Oven 645. Following hybridisation, arrays were washed and stained using the GeneChip® Fluidics Station 450 and scanned using the GeneChip® Scanner 3000 7G with Autoloader to generate a raw CEL data file for each sample.
10
Affymetrix GeneChip Clariom S Array Protocol
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RNA was converted to cDNA and biotin labelled using the WT plus kit from Affymetrix before being applied to the Affymetrix GeneChip® Human Clariom S Array (Affymetrix, Sunnyvale, CA, USA) with one array being used for each RNA sample. Following hybridization, the arrays were washed and stained on the GeneChip® Fluidics Station 450 (Affymetrix, Sunnyvale, CA, USA) using the appropriate fluidics script, before being inserted into the Affymetrix autoloader carousel and scanned using the GeneChip® Scanner 3000 (Affymetrix, Sunnyvale, CA, USA). All scanned array images were visually inspected for chip surface artefacts that could adversely impact the data.
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