The microarray study was performed as described in detail elsewhere (15 (link)–18 (link)). The total RNA isolated previously was pooled into four samples per group (control, ACTH, forskolin and adropin) treated as described above (see Cell culture and Treatment section). The protocol including in vitro transcription, biotin labeling, and cDNA fragmentation was performed using the Affymetrix GeneChip IVT Express Kit (Affymetrix, Santa Clara, CA, USA). Then the biotin labeled cDNA were hybridized with the Affymetrix Gene Chip Human Genome U219 microarrays together with appropriate internal controls. The hybridization was performed in the AccuBlockTM Digital Dry Bath hybridization oven (Labnet International, Inc., Edison, NJ, USA) at 45°C for 16 h. Subsequently, the microarrays were washed and stained by means of the Affymetrix GeneAtlas Fluidics Station (Affymetrix, Santa Clara, CA, USA). The microarrays were scanned using the Imaging Station of the GeneAtlas System. Initial analysis of the scanned microarrays was carried out with Affymetrix GeneAtlas TM Operating Software.
Genechip ivt express kit
Manufactured by Thermo Fisher Scientific
Sourced in United States
The GeneChip IVT Express Kit is a laboratory equipment product designed for the in vitro transcription of RNA from DNA templates. It provides the necessary reagents and enzymes to generate labeled complementary RNA (cRNA) from a DNA template for use in gene expression analysis applications.
Automatically generated - may contain errors
Lab products found in correlation
Geneatlas operating software, by Thermo Fisher Scientific (3 mentions)
Affymetrix geneatlas fluidics station, by Thermo Fisher Scientific (2 mentions)
Gene chip human genome u219 microarrays, by Thermo Fisher Scientific (2 mentions)
Accublock digital dry bath hybridization oven, by Labnet (2 mentions)
Genechip mouse genome 430 2.0 array, by Thermo Fisher Scientific (2 mentions)
Accublock digital dry bath, by Labnet (1 mentions)
Genechip rat genome 230 2.0 array, by Thermo Fisher Scientific (1 mentions)
Ingenuity pathway analysis software, by Qiagen (1 mentions)
Gcos software, by Thermo Fisher Scientific (1 mentions)
Fluidics station 400, by Thermo Fisher Scientific (1 mentions)
Geneatlas fluidics station, by Thermo Fisher Scientific (1 mentions)
Human genome u133 plus 2.0 microarray, by Thermo Fisher Scientific (1 mentions)
Genechip expression analysis technical manual, by Thermo Fisher Scientific (1 mentions)
Genechip scanner, by Thermo Fisher Scientific (1 mentions)
Rneasy mini kit, by Qiagen (1 mentions)
2100 bioanalyzer, by Agilent Technologies (1 mentions)
Nd 1000 spectrophotometer, by Agilent Technologies (1 mentions)
Dmem f12, by Thermo Fisher Scientific (1 mentions)
Genechip scanner 3000, by Thermo Fisher Scientific (1 mentions)
Trypsin, by Thermo Fisher Scientific (1 mentions)
10 protocols using genechip ivt express kit
1
Microarray Analysis of Gene Expression
2
Microarray Analysis of LEP-Treated Samples
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The microarray study was carried out according to the previously described procedure [69 (link),70 (link),71 (link),72 (link)]. The previously isolated RNA was pooled into four samples, representing control group (n = 2) and experimental group (n = 2) where the RNA was isolated after 24 hours from LEP administration (concentration 1 × 10−6 M). The protocol involving transcription in vitro, biotin labelling and cDNA fragmentation for further hybridization was carried out using the Affymetrix GeneChip IVT Express Kit (Affymetrix, Santa Clara, CA, USA). Obtained biotin-labelled fragments were hybridized with Affymterix GeneChip Human Genome U219 microarrays together with control cDNA and oligo B2. The hybridization process was conducted with the use of the AccuBlockTM Digital Dry Bath (Labnet International, Inc., Edison, NJ, USA) hybridization oven at 45 °C for 16 h. Then the microarrays were washed and stained according to the technical protocol using the Affymetrix GeneAtlas Fluidics Station (Affymetrix, Santa Clara, CA, USA). The array strips were scanned by the Imaging Station of GeneAtlas System. Preliminary analysis of the scanned chips was performed using Affymetrix GeneAtlasTM Operating Software. The quality of gene expression data was verified using the quality control criteria established by the software. Obtained CEL files were imported into downstream data analysis.
3
Affymetrix Gene Expression Profiling
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Complementary DNA and then biotin-labeled complementary RNA were synthesized from the
total RNA using the GeneChip® IVT Express kit (Affymetrix, Inc., Santa Clara,
CA, USA), in which an oligo-dT primer was contained, according to the manufacturer’s
instructions. Each biotin-labeled cRNA sample (approximately 10 μg) was individually
hybridized to a GeneChip® Rat Genome 230 2.0 Array (Affymetrix, Inc.) at 45°C
for 16 h, followed by washing and staining with streptavidin-phycoerythrin using the
Fluidics Station 400 (Affymetrix, Inc.). The scanned image was analyzed with the MAS5
algorithm using the GCOS software (Affymetrix, Inc.). All MAS5-analyzed data were scaled
via global normalization. Genes whose mean signal intensity values were significantly
different from those of the concurrent control with a p-value of <0.05 and whose
detection calls were not absent were analyzed using QIAGEN’s Ingenuity Pathway Analysis
(IPA) software (Qiagen, Tokyo, Japan). Three types of functional analyses using IPA were
performed: canonical pathway analysis, upstream regulator analysis, and downstream
effector analysis. In the individual gene expression analyses, genes with mean signal
intensity values at least 1.2-fold higher/0.83-fold lower than those of the concurrent
control were regarded to have significantly changed.
total RNA using the GeneChip® IVT Express kit (Affymetrix, Inc., Santa Clara,
CA, USA), in which an oligo-dT primer was contained, according to the manufacturer’s
instructions. Each biotin-labeled cRNA sample (approximately 10 μg) was individually
hybridized to a GeneChip® Rat Genome 230 2.0 Array (Affymetrix, Inc.) at 45°C
for 16 h, followed by washing and staining with streptavidin-phycoerythrin using the
Fluidics Station 400 (Affymetrix, Inc.). The scanned image was analyzed with the MAS5
algorithm using the GCOS software (Affymetrix, Inc.). All MAS5-analyzed data were scaled
via global normalization. Genes whose mean signal intensity values were significantly
different from those of the concurrent control with a p-value of <0.05 and whose
detection calls were not absent were analyzed using QIAGEN’s Ingenuity Pathway Analysis
(IPA) software (Qiagen, Tokyo, Japan). Three types of functional analyses using IPA were
performed: canonical pathway analysis, upstream regulator analysis, and downstream
effector analysis. In the individual gene expression analyses, genes with mean signal
intensity values at least 1.2-fold higher/0.83-fold lower than those of the concurrent
control were regarded to have significantly changed.
4
Affymetrix Microarray Protocol for Gene Expression
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The microarray study was performed as described in detail elsewhere [30 (link)]. The total RNA isolated from two types of cells was pooled into four samples per group (parental U2OS and ΔMVP-U2OS). The protocol including in vitro transcription, biotin labeling, and cDNA fragmentation was performed using the Affymetrix GeneChip IVT express kit (Affymetrix, Santa Clara, CA, USA). Then, the biotin labeled cDNA were hybridized with the Affymetrix Gene Chip Human Genome U219 microarrays together with appropriate internal controls. The hybridization was performed in the AccuBlockTM digital dry bath hybridization oven (Labnet International, Inc., Edison, NJ, USA) at 45 °C for 16 h. Subsequently, the microarrays were washed and stained by means of the Affymetrix GeneAtlas Fluidics Station (Affymetrix, Santa Clara, CA, USA). The microarrays were scanned using the imaging station of the GeneAtlas System. Initial analysis of the scanned microarrays was carried out with Affymetrix GeneAtlas TM Operating Software.
5
Transcriptomic Analysis of Bladder Cancer Cell Lines
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Total RNA was extracted from T24 and T24R2 cell lines using the mirVanaTM isolation kit (mirVana microRNA Isolation Kit, Ambion [Life Technologies], Austin, TX, USA) in accordance with the manufacturer's instructions. RNA samples that had high RNA integrity numbers (RIN>9.0; RIN, developed by Agilent Technologies, Santa Clara, CA, USA) and had A260/A280 absorbance ratios ranging from 1.8 to 2.1 were used for cDNA synthesis. The amplification cycles of RNA to cDNA and cDNA to biotin-labeled aRNA were performed using the GeneChip IVT Express kit (Affymetrix, Santa Clara, CA, USA). The generation of biotin-labeled aRNA by in vitro transcription, hybridization to the array and washing and scanning were performed according to the manufacturer's instructions.
6
Microarray Analysis of CNE2 Cell Lines
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Gene expression microarray analyses of CNE2-IR and CNE2 mRNAs were outsourced to CapitalBio Corporation. Affymetrix's Genechip IVT Express Kit was used for cDNA synthesis and in vitro transcription. The gene expression microarray assay was performed using 5 µg of total RNA on an Affymetrix human genome U133 plus 2.0 microarray which analyzes the expression levels of 47,000 transcripts and variants, including 38500 well-characterized human genes (Affymetrix, USA). All procedures and analyses were performed according to the protocols outlined in detail in the geneChip@ expression analysis technical manual (Affymetrix, http://www.affymetrix.com/support/downloads/manuals/expression_analysis_technical_manual.pdf ). Microarray assay was performed in duplicates, utilizing two independent sets of RNA preparations. Expression data were subsequently background corrected, normalized, and polished using robust multichip average (RMA) as previously described [32] (link). mRNA signal intensities were log2 transformed, and analyzed for differentially expressed mRNAs by using the SAM (version 3.01), and the p-values of the t-test were calculated. Differentially detected mRNA signals with ≥2.0 fold-change and the P<0.01 were considered significant. Unsupervised Hierarchical Clustering was performed for the differentially expressed mRNAs with P<0.01 using Cluster 3.0 and Java TreeView-1.1.6-win.
7
Differential Gene Expression in Huh7 Cells
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To determine the differential gene expression profiles, Huh7 cells were transfected with HBeAg-expression construct (pCMVHBeAg) or the control; we then performed expression profiling using affymetrix primeviewTM human gene expression genechips. Total RNA was extracted from cells using TRIzol reagent (Cat. No. 15596018, Thermo Fisher Scientific) according to manufacturer’s instructions. cDNA synthesis, in vitro-transcription and labelling of RNA was done using the Gene chip IVT express kit (Affymetrix, Thermo Fisher Scientific). Hybridization of labelled RNA to primeviewTM human gene expression arrays was done followed by staining and washing using Gene chip fluidics station and the arrays were scanned using Gene Chip® Scanner (Affymetrix). cDNA from two biological replicates were tested separately in the cDNA microarray. The mRNA expression data were analyzed using genespring software GX 12.1. A fold change of 1.5 times with a p value < 0.05 was used to identify differentially expressed genes. The fold change differences are presented in terms of log base 2. The RMA algorithm (robust multiarray average algorithm) was used for statistical analysis. The intra array normalization was done using quantile normalization and inter-array normalization was done by calculating median of all samples.
8
Retinal Gene Expression Profiling
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At 4 h postinfection, retinas were dissected from all eyes and were immediately frozen. Total RNAs were isolated from the frozen retinas using the Qiagen RNeasy Mini kit (Qiagen, Valenica, CA) following the manufacture’s instruction with on-column DNase treatment. RNA concentrations were measured using a Nanodrop ND-1000 Spectrophotometer and RNA quality was verified with an Agilent 2100 Bioanalyzer using an RNA Nano Chip. All RNA samples displaying no visible degradation in the Bioanalyzer analysis with two sharp ribosomal peaks were deemed acceptable for further processing. Affymetrix’s GeneChip IVT Express kit was used for cDNA synthesis and in vitro transcription. Affymetrix GeneChip Mouse Genome 430 2.0 Array was used in this study and the raw image was acquired by scanning the arrays using GeneChip scanner. Multiple files were generated and exported by Affymetrix’s software Command console. These files were used for subsequent Bioinformatics analysis. Data analysis was performed using Partek’s Genomics Suite software (Partek Inc., St. Louis, Missouri). A 5-fold change in gene expression and p < 0.05 threshold were selected as the criteria for comparative array analyses. Arrays were performed in duplicate on independently obtained RNA samples (SeqWright Genomic Services, Houston, TX).
9
Microarray Analysis of Differential Gene Expression
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Gene expression microarray analysis was performed by LC Sciences using the Affymetrix GeneChip Mouse Genome 430 2.0 array. The mouse array was used because a hamster genome array was not available, and mouse was deemed the closest match. Quality control for the integrity of total RNA was performed and then the Affymetrix's GeneChip IVT Express kit was used for cDNA synthesis and in vitro transcription. mRNA identified as differentially regulated for each clone as compared to the parental GM10115 cell line are provided in the mRNA Excel Workbook S2 (mRNA.xlsx). mRNA that were differentially expressed were analyzed using the D atabase for A nnotation, V isualization and I ntegrated D iscovery (DAVID) [30] (link), [31] (link) pathway analysis based on gene ontology GOTERM BP 3, 4, and 5. Pathways were identified using the Functional Annotation Clustering feature (high classification stringency). Three replicate arrays were performed.
10
Gastric Carcinoma Cell Line HCG-27 Protocol
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The human gastric carcinoma cell line HCG-27 was obtained from the cell bank of the Chinese Academy of Sciences. Fetal bovine serum (FBS; standard) and trypsin were purchased from Gibco (IL, USA). Dulbecco's modified Eagle's medium (DMEM), DMEM/F12, epidermal growth factor (EGF), and basic fibroblast growth factor (bFGF) were purchased from Invitrogen, while the GeneChip IVT Express Kit, GeneChip Hybridization, Wash and Stain Kit, GeneChip Hybridization Oven 640, GeneChip Fluidics Station 450, and GeneChip Scanner 3000 were obtained from Affymetrix.
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