Total RNA was extracted from tissue segments with Trizol using the protocols described elsewhere [27 (link)]. Complementary DNA (cDNA) of each sample was obtained from RNA (250 ng) of each sample by using the GeneChip® Whole Transcript Plus reagent kit (Affymetrix, Santa Clara, CA, USA) following the manufacturer protocol. The hybridization of cDNA mixture of each sample was made using labeled single-strand fragments of cDNA (3.5 μg; 41 μL) and hybridization master mix (109 μL). This cocktail was incubated at 99 °C for 5 min, decreasing the temperature to 45°C afterwards. The cocktail hybridization mix (130 μL) of each sample was then loaded on a microarray chip specific for porcine species (GeneChip® Porcine Gene 1.0 ST Array, ThermoFisher Scientific, Sweden) for incubation under rotation (60 revolutions per minute) at 45 °C for 16 h. After being incubated, the hybridized array was unloaded, washed, and stained (GeneChip® Fluidics Station 450, Affymetrix). The array chip was then scanned by using GeneChip® scanner GCS3000.
Genechip whole transcript plus reagent kit
Manufactured by Thermo Fisher Scientific
Sourced in United States
The GeneChip Whole Transcript PLUS reagent kit is a laboratory product used in gene expression analysis. The kit provides reagents and protocols for the preparation and labeling of RNA samples for microarray-based whole-transcript gene expression profiling.
Automatically generated - may contain errors
Lab products found in correlation
Gcs3000 scanner, by Thermo Fisher Scientific (23 mentions)
Genechip fluidics station 450, by Thermo Fisher Scientific (21 mentions)
Genechip command console software, by Thermo Fisher Scientific (19 mentions)
Genechip human 2.0 st array, by Thermo Fisher Scientific (11 mentions)
Agilent 2100 bioanalyzer, by Agilent Technologies (8 mentions)
Whole transcript expression array, by Thermo Fisher Scientific (6 mentions)
Genechip mouse 2.0 st array, by Thermo Fisher Scientific (6 mentions)
Genechip porcine gene 1.0 st array, by Thermo Fisher Scientific (5 mentions)
Genechip wt terminal labeling kit, by Thermo Fisher Scientific (5 mentions)
Fluidics station 450, by Thermo Fisher Scientific (5 mentions)
Genechip scanner gcs3000, by Thermo Fisher Scientific (4 mentions)
Trizol reagent, by Thermo Fisher Scientific (4 mentions)
Genechip whole transcript amplification kit, by Thermo Fisher Scientific (3 mentions)
Rneasy mini kit, by Qiagen (3 mentions)
Geneatlas fluidics station, by Thermo Fisher Scientific (2 mentions)
Human gene 2.1 st array strip, by Thermo Fisher Scientific (2 mentions)
Affymetrix human transcriptome array 2, by Thermo Fisher Scientific (1 mentions)
Dna labeling reagent, by Thermo Fisher Scientific (1 mentions)
Genechip 2.0 st array, by Thermo Fisher Scientific (1 mentions)
Genechip whole transcript terminal labeling kit, by Thermo Fisher Scientific (1 mentions)
44 protocols using genechip whole transcript plus reagent kit
1
Porcine Gene Expression Profiling
2
Affymetrix Whole Transcriptome Array Protocol
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According to the standard Affymetrix protocol, 100 ng of each RNA sample was processed using an Affymetrix GeneChip Whole Transcript PLUS Reagent kit (Affymetrix, Santa Clara, CA, USA). cRNA (15 μg) was used during the second cycle of the cDNA reaction. ss-cDNA (5.5 μg) was used for fragmentation and labeling. The fragmented cDNA was labeled by terminal deoxynucleotidyl transferase using the Affymetrix proprietary DNA Labeling reagent, which is covalently linked to biotin. Hybridization cocktails containing fragmented and labeled ss-cDNA were injected into the Affymetrix Human Transcriptome Array 2.0 (Affymetrix, Santa Clara, CA, USA). Hybridization was performed at 60 rpm for 16 h at 45°C. Following washing and staining, gene chips were scanned using the Affymetrix GeneChip Command Console software (AGCC; Affymetrix, Santa Clara, CA, USA). After the scan was completed, AGCC was used to save the imaging data and compute the probe intensity data (.cel file).
3
Porcine Transcriptome Analysis using Microarray
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Total RNA was extracted using Trizol from tissue samples of each genital tract segment and quality checked following methods previously described [34 (link)]. Equal amounts of total RNA (250 ng) from each sample were used to make cDNA using GeneChip® Whole Transcript Plus reagent kit (Affymetrix, Santa Clara, CA, USA) following the manufacturer protocol. Finally, 3.5 μg of fragmented and labelled single stranded cDNA (41 μL) were mixed with 109 μL of hybridization master mix to make a cocktail hybridization mix for a single reaction. The hybridization cocktail was then incubated first at 99 °C for 5 min, followed by a descent to 45 °C until loaded on the array chip (GeneChip® Porcine Gene 1.0 ST Array, Affymetrix Inc., 3420 Central Expressway, Santa Clara, CA 95051, USA). A total of 130 μL of the cocktail hybridization mix was loaded into each array chip and was incubated at 45 °C under 60 rotations per min, for 16 h. The hybridized cartridge array chip was then unloaded and subjected to washing and staining using a GeneChip® Fluidics Station 450 (Affymetrix), to be finally scanned using the Affymetrix GeneChip® scanner GCS3000.
4
Transcriptional Profiling of Muscle Aging
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Each WAT from young sedentary (n = 4), old sedentary (n = 4), and old exercise (n = 4) groups were pooled and RNA purity and integrity were confirmed by ND-1000 Spectrophotometer (Nano-Drop, Wilmington, USA). The Affymetrix Whole transcript Expression array process was performed according to the protocol of the manufacturer (Gene Chip Whole Transcript PLUS reagent Kit). cDNA was synthesized according to the manufacturer’s method using Gene Chip Whole Transcript (WT) Amplification kit, and the hybridized array was scanned using a GCS3000 Scanner (Affymetrix). Signal values were calculated using Affymetrix® Gene ChipTM Command Console software. Gene enrichment and functional annotation analysis were performed on genes showing significant expression level (≥ 2-fold) based on gene ontology and KEGG pathway analysis, respectively. Gene Ontology Enrichment analysis was performed using g: Profiler tool (https://biit.cs.ut.ee/gprofiler/ ). The gene search procedure used the functional annotation category of REVIGO, uploaded the gene list, and selected an identifier from the official gene symbol. Species and background were selected for Mus musculus, and microarray data of this study were verified and compared.
5
Transcriptomic Analysis of β-Catenin Mutants
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RNA was extracted from the indicated cells expressing mock, wild-type, phosphomimetic (S311D), or non-phosphomimetic β-catenin (S311A). RNA purity and integrity were evaluated using an ND-1000 spectrophotometer (Nanodrop, Wilmington, DE, USA) and Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA, USA). The Affymetrix whole transcript expression array process was conducted according to the manufacturer's protocol (GeneChip Whole Transcript PLUS reagent kit). cDNA was synthesized using a GeneChip WT (Whole Transcript) Amplification kit as described by the manufacturer. The sense cDNA was then fragmented and biotin-labeled with terminal deoxynucleotidyl transferase using a GeneChip WT Terminal labeling kit. Approximately 5.5 μg of labeled DNA target was hybridized to the Affymetrix GeneChip Human 2.0 ST Array at 45°C for 16 hours. Hybridized arrays were washed and stained on a GeneChip Fluidics Station 450 and scanned on a GCS3000 Scanner (Affymetrix). Signal values were computed using Affymetrix GeneChip Command Console software.
6
Affymetrix Whole-Transcript Expression in Rat Muscle
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The Affymetrix Whole-transcript Expression array was performed according to the manufacturer's protocol (GeneChip Whole Transcript PLUS reagent kit; Affymetrix, Santa Clara, CA, USA) in rat sub-trapezial muscle. The sense complementary DNA (cDNA) was then fragmented and biotin-labeled with terminal deoxynucleotidyl transferase using the GeneChip WT Terminal labeling kit (Affymetrix). Approximately 5.5 µg of labeled DNA target was hybridized to the Affymetrix GeneChip Rat 2.0 ST Array (Affymetrix) at 45℃ for 16 h. Hybridized arrays were washed and stained on a GeneChip Fluidics Station 450 and scanned on a GCS3000 Scanner (Affymetrix). Signal values were computed using the Affymetrix® GeneChip™ Command Console software (Affymetrix) [38 (link)]. The pathway analysis of differentially expressed genes (DEGs) was based on Kyoto Encyclopedia of Genes and Genomes (KEGG) and Gene ontology (GO) databases.
7
Microarray Analysis of Radiation-Induced Transcriptome
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Stable cells were irradiated with 10 Gy radiation. After 6 h, RNA was extracted, using the TRIzol reagent. Reverse transcription was carried out, and the Affymetrix Whole Transcript Expression array was used, according to the manufacturer’s instructions, for microarray analysis (GeneChip Whole Transcript PLUS reagent kit and GeneChip Whole Transcript Amplification kit). The cDNA labeling (GeneChip WT Terminal labeling kit), hybridization (Affymetrix GeneChip 2.0 ST Array), and slide scanning (Affymetrix, GCS3000 Scanner) were performed according to the manufacturers’ instructions.
8
Affymetrix GeneChip Transcriptome Analysis
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RNA amplification, labeling, array hybridization, and scanning were conducted by Macrogen Inc. (Seoul, Korea). The Affymetrix Whole Transcript Expression array process was conducted according to the manufacturer’s protocols using a GeneChip Whole Transcript PLUS reagent kit (Affymetrix, USA). Briefly, the GeneChip Whole Transcript Amplification kit (Affymetrix) was used for cDNA synthesis. The sense cDNA was then fragmented and biotin-labeled with terminal deoxynucleotidyl transferase, using a GeneChip Whole Transcript Terminal labeling kit (Affymetrix). Approximately 5.5 μg of labeled DNA target was hybridized to the Affymetrix GeneChip Mouse 2.0 ST Array for 16 h at 45 °C, which covers more than 35,000 transcripts. After washing step, hybridized arrays were stained on a GeneChip Fluidics Station 450 and scanned on a GCS3000 Scanner (Affymetrix). Signal values were computed using the Affymetrix® GenChip™ Command Console software (AGCC).
9
Affymetrix Whole Transcript Expression Analysis
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The Affymetrix Whole transcript Expression Array process was executed according to the manufacturer's protocol (GeneChip Whole Transcript PLUS Reagent Kit; Affymetrix, Santa Clara, CA, USA). cDNA was synthesized using the GeneChip WT (whole transcript) Amplification Kit (Affymetrix) as described by the manufacturer. The sense cDNA was then fragmented and biotin-labeled with terminal deoxynucleotidyl transferase (TdT) using the GeneChip WT Terminal Labeling Kit (Affymetrix). Approximately 5.5 µg of labeled DNA target was hybridized to the Affymetrix GeneChip Human 2.0 ST Array at 45℃ for 16 hours. Hybridized arrays were washed and stained on a GeneChip Fluidics Station 450 and scanned on a GCS3000 Scanner (Affymetrix). Signal values were computed using the Affymetrix GeneChip Command Console software.
10
Transcriptomic Analysis of Mouse Tissues
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Total RNA was extracted from frozen tissues at week 20. RNA purity and integrity were evaluated by ND-1000 Spectrophotometer (NanoDrop, Wilmington, USA), and Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, USA). The Affymetrix Whole transcript Expression array process was executed according to the manufacturer’s protocol (GeneChip Whole Transcript PLUS reagent Kit). Briefly, cDNA was synthesized using the GeneChip WT (Whole Transcript) Amplification kit as described by the manufacturer. The sense cDNA was then fragmented and biotin-labeled with TdT (terminal deoxynucleotidyl transferase) using the GeneChip WT Terminal labeling kit. Approximately 5.5 μg of labeled DNA target was hybridized to the Affymetrix GeneChip Mouse Array at 45 °C for 16 h. Hybridized arrays were washed and stained on a GeneChip Fluidics Station 450 and scanned on a GCS3000 Scanner (Affymetrix). Signal values were computed using the Affymetrix® GeneChip™ Command Console software. The assay was performed by the Macrogen (Korea).
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