Per offspring, colon and SI samples were analysed as described previously [43 (link)], and in total, six male and six female samples were used in this analysis (three of the nine females were not included in the microarray analysis due to budget limitations). In brief, 100 ng of purified RNA was used for the preparation of labelled cDNA, applying the Ambion Whole Transcript (WT) Expression Kit (Life Technologies, Carlsbad, CA, USA) in combination with the Affymetrix GeneChip WT Terminal Labeling Kit (Affymetrix, Santa Clara, CA, USA). All samples were hybridized at one time point to Affymetrix GeneChip Mouse Gene 1.1 ST arrays according to standard Affymetrix protocols. Quality control and normalization were performed using Bioconductor software packages integrated in an on-line pipeline [44 (link)]. Normalized expression estimates of probe sets were computed by the robust multiarray (RMA) analysis algorithm available in the Bioconductor library AffyPLM using default settings [45 (link)]. Probe sets were redefined according to Dai et al. [46 (link)] and assigned to unique gene identifiers (IDs) of the Entrez Gene database, resulting in 21,187 assigned Entrez IDs. Array data were submitted to the Gene Expression Omnibus and are available under accession number GSE57516.
Ambion whole transcript expression kit
Manufactured by Thermo Fisher Scientific
Sourced in United States, Canada
The Ambion Whole Transcript (WT) Expression Kit is a laboratory product designed for gene expression analysis. It is used to generate amplified and labeled complementary DNA (cDNA) from total RNA samples. The kit provides reagents and protocols for the entire process, from RNA input to labeled cDNA output, suitable for downstream applications such as microarray analysis.
Automatically generated - may contain errors
Lab products found in correlation
Genetitan system, by Thermo Fisher Scientific (3 mentions)
Rneasy mini kit, by Qiagen (3 mentions)
Genechip wt terminal labeling kit, by Thermo Fisher Scientific (2 mentions)
Affymetrix genechip wt terminal labeling kit, by Thermo Fisher Scientific (2 mentions)
Affymetrix genechip mouse gene 1.1 st arrays, by Thermo Fisher Scientific (2 mentions)
Rat gene 1.1 st 16 array plate, by Thermo Fisher Scientific (2 mentions)
Genechip scanner 3000 7g system, by Thermo Fisher Scientific (2 mentions)
Affymetrix wt sense target labeling kit, by Thermo Fisher Scientific (2 mentions)
Genechip mouse transcriptome assay 1.0 st arrays, by Thermo Fisher Scientific (2 mentions)
Fs450 fluidic station, by Thermo Fisher Scientific (2 mentions)
Trizol reagent, by Thermo Fisher Scientific (2 mentions)
Nanodrop 2000, by Thermo Fisher Scientific (2 mentions)
Genechip mouse gene 1.1 st arrays, by Thermo Fisher Scientific (1 mentions)
Genetitan multi channel instrument, by Thermo Fisher Scientific (1 mentions)
Bioanalyzer, by Agilent Technologies (1 mentions)
Affymetrix expression console software, by Thermo Fisher Scientific (1 mentions)
Genechip scanner 3000, by Thermo Fisher Scientific (1 mentions)
Rnalater, by Thermo Fisher Scientific (1 mentions)
14 protocols using ambion whole transcript expression kit
1
Mouse Whole Transcriptome Expression Profiling
2
Rapamycin response in miR-150 transfected Jurkat cells
3
Microarray Analysis of Liver RNA Samples
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Hundred nanograms of purified RNA from the liver of the individual mice was used for the preparation of labeled cDNA, applying the Ambion Whole Transcript (WT) Expression kit (Life Technologies, Carlsbad, USA) in combination with the Affymetrix GeneChip WT Terminal Labeling kit (Affymetrix, Santa Clara, USA). All samples were hybridized at one time point to Affymetrix GeneChip Mouse Gene 1.1 ST arrays according to standard Affymetrix protocols. Microarray analysis was performed in MADMAX, a pipeline for statistical analysis of microarray data 42 . Arrays were normalized using the Robust Multiarray Average 43 , 44 . Probe sets were defined according to Dai et al. 45 . In this method probes are assigned to unique gene identifiers, in this case Entrez IDs. The probes on the Gene 1.1 ST arrays represent 21 225 Entrez IDs. Array data were submitted to the Gene Expression Omnibus and are available under accession number GSE93642.
4
Affymetrix Gene Expression Profiling
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We prepared cDNA from total RNA using the Ambion Whole Transcript (WT) Expression Kit (Ambion) as previously described [15 (link)]. The cDNA was fragmented, labeled, hybridized, stained, and washed per the manufacturer’s recommendations (Affymetrix, Santa Clara, CA). Samples were then applied to the Rat Gene 1.1 ST 16 Array Plate or 24 Array Plate and placed in the GeneTitan System (Affymetrix) according the manufacturer’s recommendations. Of the 144 arrays, 2 liver arrays, 1 lung array, and 1 heart array did not pass quality control checks and were excluded from further analysis.
5
Rat Gene Expression Profiling
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RNA was diluted to 50 ng/μL, and synthesized to cRNA using the Ambion Whole Transcript (WT) Expression Kit (Ambion, Cat. No. 4411974, Austin, TX) according to the manufacturer’s instructions. The cRNA was quantified by NanoDrop and 455 ng/μL was used as a template to synthesize cDNA. The samples were then fragmented using the GeneChip WT Terminal Labeling Kit (Cat. No. 901524, Affymetrix, Santa Clara, CA), labeled, and prepared with the GeneTitan Hybridization, Wash and Stain Kit for whole-transcript array plates (Affymetrix, Cat. No. 901622), according to the manufacturer’s instructions. Samples were then applied to the Rat Gene 1.1 ST 16 Array Plate or 24 Array Plate (Affyemetrix, Cat. No. 901432, 901431, respectively) and placed in the GeneTitan System (Affymetrix, Cat. No. D0101330), according to the manufacturer’s recommendations. Of the 44 arrays, two did not hybridize appropriately and did not pass the GeneTitan scanning QC, and were excluded from further analysis.
6
Rat Gene 1.1 ST Array Protocol
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We prepared cDNA from total tissue RNA using the Ambion Whole Transcript (WT) Expression Kit (Ambion) according to manufacturer’s recommendations, fragmenting the cDNA using the GeneChip WT Terminal Labeling Kit (Affymetrix, Santa Clara, CA). Following manufacturer’s instructions, we prepared labeled cDNA, trays, and arrays with the GeneTitan Hybridization, Wash and Stain Kit for WT array plates (Affymetrix). Samples were then applied to the Rat Gene 1.1 ST 16 Array Plate or 24 Array Plate and placed in the GeneTitan System (Affymetrix), according manufacturer’s recommendations. Of the 144 arrays, 2 liver, 1 lung, and 1 heart array did not pass the GeneTitan scanning quality control. Thus, 4 arrays (2.7%) were excluded from further analysis.
7
Microarray Analysis of Mouse Brain Transcriptome
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The Affymetrix Genechip Mouse Clariom S was used to examine gene expression (Affymetrix, Santa Clara, CA, USA). Sample processing and generation of Microarray data was completed by experienced technicians in the Molecular Resource Center at UTHSC. In brief, two hundred nanograms of DNase-treated total RNA was amplified, labeled, and fragmented using Ambion Whole Transcript (WT) Expression Kit according to the manufacturer’s protocol (Thermo Fisher Scientific, Santa Clara, CA USA). Samples were hybridized overnight according to manufacturer’s protocols; samples were then washed and stained on Affymetrix GeneChip Fluidics Station 450 (Affymetrix, Santa Clara, CA, USA) followed by scanning on the GeneChip Scanner 3000 (Applied Biosystems, Waltham, MA, USA). Data was normalized and analyzed for quality control in Affymetrix Expression Console Software using RMA-sketch normalization (Affymetrix, Santa Clara, CA, USA). After normalization and quality control, a total number of 22,203 probe sets were used for subsequent data analysis. A total of 128 samples were used—4 samples per treatment (control, ethanol), per sex (male, female), and per strain (B6, D2, BXD2, BXD48a, BXD60, BXD71, BXD73, BXD100) (Supplemental Table S1 ).
8
Transcriptional Profiling of LCLs in ALS
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Transcriptional profiling of peripheral LCL’s from ALS patients with short and long disease was performed using Affymetrix Human Exon 1.0ST GeneChip microarrays. In brief, ∼300 ng of extracted RNA was processed using the Ambion Whole Transcript (WT) Expression Kit (Thermo Fisher Scientific) producing fragmented biotin-labelled sense-stranded copy DNA that was hybridized onto Human Exon 1.0ST GeneChip microarrays. Post-hybridization array washing, and staining were performed in the GeneChip Fluidics station and arrays scanned using the GeneChip 3000 7G scanner.
9
Colonic Transcriptome Profiling via Microarray
10
Colonic Transcriptome Profiling via Microarray
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We used a 0.5 cm segment of the proximal colon for analysis. RNA was extracted using TRIZOL reagent (Life Technologies, Gaithersburg, MD) and RNeasy mini kit (QIAGEN, Valencia, CA). Total RNA (100 ng) was amplified and labeled using the Ambion whole-transcript expression kit (Life Technologies) and Affymetrix WT sense target labeling kit (Affymetrix, Santa Clara, CA). Affymetrix GeneChip Mouse Transcriptome Assay 1.0 ST arrays were hybridized with 540 ng of labeled sense DNA, washed, and stained using the FS450 Fluidic Station (Affymetrix). Fluorescent signal intensities of each stained chip were captured by the GeneChip Scanner 3000 7G System (Affymetrix).
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