The same amounts of RNA from different animals in each group were pooled (n = 5) as one microarray sample and used for poly A tailing and ligation with the FlashTag Biotin HSR RNA Labeling Kit (Affymetrix, Santa Clara, CA, USA). The labeled samples were mixed with a hybridization cocktail (2×Hybridization Mix, 27.5% formamide, DMSO, 20× Eukaryotic Hybridization Controls, Control Oligonucleotide B2 from GeneChip Eukaryotic Hyb Control Kit, Affymetrix) and hybridized onto an miRNA 2.0 Array (Affymetrix) at 48 °C for 16 h. Thereafter, washing and staining were performed using an Affymetrix GeneChip Fluidics Station 450. Scans were performed using an Affymetrix GeneChip Scanner 3000. Data analysis was performed using the Affymetrix miRNA Array QC tool. Each experiment was performed following the manufacturer’s instructions.
Eukaryotic hybridization controls
Manufactured by Thermo Fisher Scientific
The Eukaryotic Hybridization Controls are a set of nucleic acid controls designed for use with eukaryotic gene expression analysis platforms. The controls provide a reference for monitoring the performance and consistency of the hybridization process.
Automatically generated - may contain errors
Lab products found in correlation
Flashtag biotin hsr rna labeling kit, by Thermo Fisher Scientific (1 mentions)
Mirna 2.0 array, by Thermo Fisher Scientific (1 mentions)
Genechip fluidics station 450, by Thermo Fisher Scientific (1 mentions)
Genechip scanner 3000, by Thermo Fisher Scientific (1 mentions)
Microarray suite 5, by Thermo Fisher Scientific (1 mentions)
Streptavidin phycoerythrin, by Thermo Fisher Scientific (1 mentions)
2 protocols using eukaryotic hybridization controls
1
miRNA Profiling of Animal Samples
2
Comparative Microarray Hybridization Protocols
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Example 4
NZ tumours: Hybridisation of the labelled target cDNA was performed using MWG Human 30K Array oligonucleotides printed on epoxy coated slides. Slides were blocked with 1% BSA and the hybridisation was done in pre-hybridisation buffer at 42° C. for at least 12 hours followed by a high stringency wash. Slides were scanned with a GenePix Microarray Scanner and data was analyzed using GenePix Pro 4.1 Microarray Acquisition and Analysis Software (Axon, CA).
DE tumours: cRNA was mixed with B2-control oligonucleotide (Affymetrix, Santa Clara, Calif.), eukaryotic hybridization controls (Affymetrix, Santa Clara, Calif.), herring sperm (Promega, Madison, Wis.), buffer and BSA to a final volume of 300 μl and hybridized to one microarray chip (Affymetrix, Santa Clara, Calif.) for 16 hours at 45° C. Washing steps and incubation with streptavidin (Roche, Mannheim, Germany), biotinylated goat-anti streptavidin antibody (Serva, Heidelberg, Germany), goat-IgG (Sigma, Taufkirchen, Germany), and streptavidin-phycoerythrin (Molecular Probes, Leiden, Netherlands) was performed in an Affymetrix Fluidics Station according to the manufacturer's protocol. The arrays were then scanned with a HP-argon-ion laser confocal microscope and the digitized image data were processed using the Affymetrix® Microarray Suite 5.0 Software.
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