5μg total RNA was used to synthesize first strand cDNA using oligonucleotide probes with 24 oligo-dT plus T7 promoter (Proligo LLC, St. Louis, MO), and the SuperScript Choice System (Invitrogen). cRNA probe was generated and biotinylated using the BioArray RNA High Yield Transcript Labeling kit (ENZO Life Sciences Inc). Fragmented cRNA (10μg) was hybridized to the GeneChip Human Exon 1.0 ST Array (Affymetrix, Inc, Santa Clara, CA) for 16h at 45°C, and incubated with streptavidin-phycoerythrin conjugate. Staining was amplified with biotinylated goat anti-streptavidin antibody, followed by staining with a streptavidin-phycoerythrin conjugate. Fluorescence was detected using the Affymetrix G3000 GeneArray Scanner and GeneChips were analyzed with the GeneChip Operating System 1.1.1 (GCOS) software from Affymetrix.
G3000 genearray scanner
Manufactured by Thermo Fisher Scientific
The G3000 GeneArray Scanner is a high-performance microarray scanner designed for gene expression analysis. It utilizes a laser and photomultiplier tube detection system to precisely measure the fluorescence intensity of probes on microarray slides. The scanner supports a wide range of microarray formats and provides users with reliable, quantitative data for their research and applications.
Automatically generated - may contain errors
Lab products found in correlation
Fluidics station 450, by Thermo Fisher Scientific (4 mentions)
Flashtag biotin rna labeling kit for affymetrix genechip mirna arrays, by Thermo Fisher Scientific (2 mentions)
Expression console, by Thermo Fisher Scientific (2 mentions)
Affymetrix genechip mirna array, by Thermo Fisher Scientific (2 mentions)
Genechip mirna 3.0 array, by Thermo Fisher Scientific (2 mentions)
Genechip mirna array, by Thermo Fisher Scientific (2 mentions)
Superscript choice system, by Thermo Fisher Scientific (1 mentions)
Genechip human exon 1.0 st array, by Thermo Fisher Scientific (1 mentions)
Gene chip mirna 4.0 microarray, by Thermo Fisher Scientific (1 mentions)
Jmp genomics 6, by SAS Institute (1 mentions)
Flashtag biotin rna labelling kit, by Thermo Fisher Scientific (1 mentions)
Wt expression kit, by Thermo Fisher Scientific (1 mentions)
Genechip sample cleanup module, by Thermo Fisher Scientific (1 mentions)
Rneasy micro, by Qiagen (1 mentions)
2100 bioanalyzer, by Agilent Technologies (1 mentions)
7 protocols using g3000 genearray scanner
1
Transcriptome Analysis Using Affymetrix Exon Array
2
Affymetrix GeneChip miRNA Profiling of Mutant p53 Cells
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The H1299 cells stably transfected with mutp53 R282W or the control vector were respectively analyzed by Affymetrix GeneChip miRNA 4.0 microarray with three biological replications. A total of 200 ng small RNA was used in sample preparation with a FlashTag Biotin RNA Labeling Kit for Affymetrix GeneChip miRNA arrays (Genisphere). The labeled RNA was consequently hybridized for sixteen hours to an Affymetrix GeneChip miRNA array according to the product manual. Microarrays were washed and stained in the Affymetrix Fluidics Station 450, and scanned on the Affymetrix G3000 GeneArray Scanner. The image files were analyzed using the Affymetrix software (Expression Console), Robust Multi-array Average (RMA) background correction, log-2 transformations and global normalization methods were performed for data pre-processing, and normalization.
3
Porcine miRNA Expression Profiling
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The Affymetrix GeneChip miRNA 3.0 Array (Affymetrix) was used to determine the miRNA expression profile of the LM 24 h ante mortem of Duroc and PiNN pigs. It is comprised of 16,772 entries representing hairpin precursor, total probe set 19,724 for detection of most miRNA from 153 species (miRBase V.17). 200 ng of small RNA were used in sample preparation using a FlashTag Biotin HSR RNA Labeling kit (Genisphere) for Affymetrix GeneChip miRNA arrays. Each of the labeled RNA (n = 20) was then hybridized for 16 h to an Affymetrix GeneChip miRNA array according to manufacturer’s recommendation (Affymetrix), washed and stained in the Affymetrix Fluidics Station 450, and scanned on the Affymetrix G3000 Gene Array Scanner. Expression Console software was used for robust multichip average (RMA) normalization and the detection of present miRNAs by applying the DABG (detection above background) algorithm. Further filtering was done by excluding probes that were present in less than 70 % of the samples within each breed and annotated miRNAs that had sequence greater than or equal to 30 nt in length. Three thousand five hundred eighty seven probes passed the quality filtering and were used for further analysis. The availability of expression data are in the Gene Expression Omnibus public repository with the GEO accession number GSE80198: GSM2120718-GSM2120737.
4
MicroRNA Profiling in C2C12 Myoblasts
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MicroRNA expression profiling was performed using Affymetrix Gene Chip Micro 3.0 Array (Affymetrix, Inc, Santa Clara, CA, USA) containing 16,772 entries representing hairpin precursor (miRBase v17) (in total 19,724 probe sets for detection most of miRNA from 153 species), which provides >3-log dynamic range, with >95% reproducibility and 85% transcript detection at 1.0 amol, for a total RNA input of 130–500 ng. A total of 9 enriched small-RNA pools derived from D0, D4, and D8 post-induction of C2C12 myoblasts (three each) were used in the array hybridizations. Each RNA pool was generated from 5 individual RNA samples extracted from independent cultures. 200 ng of small RNA were used in sample preparation with a FlashTag Biotin RNA Labeling Kit for Affymetrix GeneChip miRNA arrays (Genisphere). The labeled RNA was then hybridized for 16 hours to an Affymetrix GeneChip miRNA array according to the manufacturer’s recommendations (Affymetrix), washed and stained in the Affymetrix Fluidics Station 450, and scanned on the Affymetrix G3000 GeneArray Scanner. The image files were analyzed using the Affymetrix software (Expression Console), Robust Multi-array Average (RMA) background correction, log-2 transformations and quantile normalization methods implemented in JMP Genomics 5.1 were performed for data pre-processing, normalization, and statistical analysis.
5
Bovine miRNA Expression Profiling
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We used the GeneChip miRNA 3.0 array (Affymetrix, Santa Clara, CA, USA) for miRNA expression profiling essentially as described by the manufacturer. This array included all microRNAs listed in release 17 of miRBase. The microarray is multispecies, covering almost 20,000 miRNAs, including 676 mature miRNAs assigned to Bos taurus. Labelling of small RNAs (200 ng/sample) was done with a FlashTag Biotin RNA labelling kit for Affymetrix GeneChip miRNA arrays (Genisphere, Hatfield, PA, USA). Hybridzation of the labelled small RNA with the array lasted 16 h. Subsequent washing and staining were performed in a Fluidics Station 450, and the arrays were then scanned on a G3000 GeneArray Scanner (Affymetrix). Robust multi-array average (RMA) background correction, log2 transformations and quantile normalization methods implemented in JMP Genomics 6 (SAS Institute, Cary, NC, USA) were applied.
6
Affymetrix miRNA Microarray Analysis
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Small RNA fractions (200 ng) were used for sample preparation using a FlashTag BioTin RNA labelling kit (Affymetrix, Santa Clara, CA, USA). Fragmented biotin-labelled cRNAs were further hybridized for 16 hours to an Affymetrix GeneChip miRNA 3.0 Array containing 19,724 probe-sets designed from 153 species based on miRBase version 17. After staining and washing on an Affymetrix Fluidics Station 450, arrays were scanned on an Affymetrix G3000 Gene Array Scanner. Raw data were pre-processed using Affymetrix GCOS 1.1.1 software.
7
Thymic RNA Profiling in Pre-diabetic Mice
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RNA was obtained from the thymi of pre-diabetic treated mice at 4 and 6 weeks of treatment, using RNeasy Micro (QIAGEN, Hilden, Germany). Thymic glands were also obtained from a NOD mouse control group for each time point. RNA quality (2100 Bioanalyzer, Agilent Technologies Inc., Santa Clara, CA) was optimal for microarray experiments (RIN between 6 and 8). cDNA was synthesized with 50-100 ng of total RNA using the WT expression kit (Ambion, Applied Biosystems, CA, USA), fragmented and labeled with the Terminal labeling kit (Affymetrix, Inc. Santa Clara, CA), purified (GeneChip® Sample Cleanup Module, Affymetrix), fragmented and checked to verify its integrity. Mouse Gene1.1 ST 16 array plates (28.853 genes) were hybridized and scanned by an Affymetrix G3000 Gene Array Scanner.
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