Prior to hybridization, the fragmented and biotin labelled cRNA from each rat ovary was mixed with control oligonucleotide B2 (3 nM), 20× eukaryotic hybridization controls (bioB, bioC, bioD, cre) (Affymetrix, CA, USA), 2× hybridization mix and DMSO. The hybridization cocktail were then incubated at 99°C (5 min) and subsequently at 45°C (5 min). Each sample was then transferred to independent GeneChip® Rat Genome 230 2.0 Array chip. Three biological replicates and one technical replicate (pool of three biological replicates) were hybridized for each rat group for 16 h. The array slides were washed and stained using the Fluidics Station 450/250 (Affymetrix, CA, USA), according to the GeneChip® expression user manual (P/N 702232 Rev. 3). Arrays were scanned with the GeneChip™ 3000 laser confocal slide scanner (Affymetrix, CA, USA) integrated with GeneChip® Operating System (GCOS).
Fluidics station 450 250
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Fluidics Station 450/250 is a laboratory instrument designed for automated sample preparation and processing. It is capable of performing various fluid handling tasks, such as sample mixing, dilution, and reagent addition, with precision and consistency. The core function of the Fluidics Station is to automate these liquid handling processes, thereby improving efficiency and reproducibility in a variety of laboratory applications.
Automatically generated - may contain errors
Lab products found in correlation
Genechip 3000 laser confocal slide scanner, by Thermo Fisher Scientific (2 mentions)
Genechip primeview human gene expression array, by Thermo Fisher Scientific (1 mentions)
Genechip command console, by Thermo Fisher Scientific (1 mentions)
3000 7g plus instrument, by Thermo Fisher Scientific (1 mentions)
Genechip bovine genome array, by Thermo Fisher Scientific (1 mentions)
Genechip 3 ivt express kit, by Thermo Fisher Scientific (1 mentions)
Transcription analysis console, by Thermo Fisher Scientific (1 mentions)
Expression console, by Thermo Fisher Scientific (1 mentions)
Genechiptm wt plus reagent kit, by Thermo Fisher Scientific (1 mentions)
Genechip scanner 3000, by Thermo Fisher Scientific (1 mentions)
Genechip operating software, by Thermo Fisher Scientific (1 mentions)
Genearray scanner, by Thermo Fisher Scientific (1 mentions)
Genechip wt terminal labeling kit, by Thermo Fisher Scientific (1 mentions)
Bionalyzer, by Agilent Technologies (1 mentions)
Rneasy mini kit, by Qiagen (1 mentions)
5 protocols using fluidics station 450 250
1
Rat Genome Array-Based Transcriptome Analysis
2
Gene Expression Profiling of Human Samples
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Gene expression profiling was performed using an Affymetrix Gene Chip® PrimeView™ Human Gene Expression Array, which contains 36,000 probe sets representing 20,000 well-characterized human genes (Affymetrix, Santa Clara, CA). Total RNA was isolated from duplicate cultures using the TRIzol method. After passing a quality control measurement, RNA was amplified and labeled using the Affymetrix 3ʹ IVT Express kit and hybridized to the array for 16 h. This was followed by washing and staining using the Affymetrix Fluidics Station 450/250. Arrays were scanned using an Affymetrix 3000 7G plus instrument. Affymetrix GeneChip Command Console (AGCC) software was used to generate.cel files. Data were imported into Agilent Gene Spring GX software for further analysis. Unsupervised hierarchical clustering analysis was performed using Cluster 3.0 software (Stanford University, USA). The row dendrogram was generated using Ward’s clustering method with a half square Euclidean distance measure. The column dendrogram was generated using the single linkage clustering method and a Euclidean distance measure. Gene ontology analysis was based on David 6.7 (http://david.abcc.ncifcrf.gov/home.jsp
The transcriptome data have been deposited in the NCBI Gene Expression Omnibus and are accessible through GEO Series accession number GSE145786.
The transcriptome data have been deposited in the NCBI Gene Expression Omnibus and are accessible through GEO Series accession number GSE145786.
3
Transcriptomic Analysis of Bovine Tissues
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The total RNA samples isolated from each SE, CE and HE in three biological replicates were subjected to gene expression analysis using the GeneChip bovine Genome array (Affymetrix, CA, USA). For this, we performed RNA amplification, cDNA synthesis, labelling and array hybridization for each of the three cows of SE, CE or HE group according to the recommendations and suggestion of the GeneChip®3′ IVT Express Kit (Affymetrix, CA, USA). Three hybridizations were preformed for each SE, CE or HE animals and the three hybridization represented the biological replicates correspond to three animals of SE, CE or HE group. The array slides were then washed and stained using the Fluidics Station 450/250 (Affymetrix, CA, USA) following the GeneChip® expression wash, stain and scan user manual. After 16 h of hybridization, the arrays were scanned with the GeneChip™3000 laser confocal slide scanner (Affymetrix, CA, USA) integrated with GeneChip® Operating System (GCOS). The signal intensity of the control probes were monitored during array scanning
4
Transcriptome Analysis of NRAS Knockdown
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mRNA expression in siRNA- (siNRAS#1) or control-transfected hPheo1 cells was analysed with the GeneChip HuGene ST 1.0 array, GeneChipTM WT PLUS Reagent Kit, Fluidics Station 450/250, GeneChip® Hybridization Wash and Stain Kit, and GeneChip® Scanner 3000 (Affymetrix, Santa Clara, CA, USA). CEL files were quality-checked with Expression Console (Thermo Fisher; analysis settings: RMA, “gene level”), and CHP files analysed with Transcription Analysis Console (Thermo Fisher). Genes with p values < 0.05 (ANOVA), FDR-corrected p values < 0.25, and fold change < −1.5 or >1.5 were considered significantly regulated genes. Gene set overlaps were computed using the Molecular Signatures database webpage (MSigDB v6.2) [27 (link)] for the “Hallmark” [28 (link)] and “C2: Canonical pathways” [27 (link)] gene sets, with p values calculated from the hypergeometric distribution and corrected for multiple testing with the Benjamini and Hochberg procedure. Heatmaps displaying the expression of genes of interest were generated with Wolfram Mathematica version 11.1 (Wolfram Research, Champaign, IL, USA).
5
RNA Isolation and Microarray Analysis
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RNA from the HepaRG cells was prepared using the RNeasy mini kit from Qiagen (France) as described previously [55] except that a DNase I step was included in the protocol.
For the microarray studies, the quality of the RNA (RIN value) was assessed with a Bionalyzer (Agilent Technologies) [56] .
ssDNA (sense single stranded DNA) was synthesized using the Affymetrix GeneChip Whole Transcript Sense Target Labelling Assay kit, according to the manufacturer's protocol. ssDNA samples were then fragmented according to the Affymetrix protocol. The purified ssDNA was quantified and its quality was assessed with a Bioanalyzer. Subsequent labeling of the samples was performed by synthesis of Biotin-labeled ssDNA using the GeneChip WT Terminal Labeling kit (Affymetrix). ssDNA targets were hybridized onto high-density microarrays (Affymetrix Human Genome 1.0 ST GeneChip array) according to the Affymetrix Eukaryotic Target manual. The microarrays were then washed and stained using the Affymetrix fluidics station 450/250 and Genechip Operating Software and scanned with an Affymetrix GeneArray scanner. The raw affymetrix datasets (.CEL) are available in the Gene Expression Omnibus database (http://www.ncbi.nlm.nih.gov/geo/) with the accession number (GSE46874).
For the microarray studies, the quality of the RNA (RIN value) was assessed with a Bionalyzer (Agilent Technologies) [56] .
ssDNA (sense single stranded DNA) was synthesized using the Affymetrix GeneChip Whole Transcript Sense Target Labelling Assay kit, according to the manufacturer's protocol. ssDNA samples were then fragmented according to the Affymetrix protocol. The purified ssDNA was quantified and its quality was assessed with a Bioanalyzer. Subsequent labeling of the samples was performed by synthesis of Biotin-labeled ssDNA using the GeneChip WT Terminal Labeling kit (Affymetrix). ssDNA targets were hybridized onto high-density microarrays (Affymetrix Human Genome 1.0 ST GeneChip array) according to the Affymetrix Eukaryotic Target manual. The microarrays were then washed and stained using the Affymetrix fluidics station 450/250 and Genechip Operating Software and scanned with an Affymetrix GeneArray scanner. The raw affymetrix datasets (.CEL) are available in the Gene Expression Omnibus database (http://www.ncbi.nlm.nih.gov/geo/) with the accession number (GSE46874).
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