Protocols
Affymetrix GeneChip
Labeling of Target With Biotin: The laboratory uses the Affymetrix Eukaryotic One-Cycle (2 to 10 μg of total RNA) and Two-Cycle (10 to 100 ng of total RNA) Target Labeling Assays for generating biotin-labeled samples for hybridization to Affymetrix GeneChips. The Affymetrix Two-Cycle Target Labeling Assay uses an additional cycle of cDNA synthesis and IVT amplification to obtain sufficient amounts of labeled cRNA target.
Hybridization: The GeneChip arrays are hybridized overnight in the Affymetrix hybridization oven at 42oC.
Washing and Staining: The laboratory uses the Affymetrix GeneChip Fluidics Station 450 to perform the automated Affymetrix wash and stain protocols.
Scanning: The Affymetrix GeneChip Scanner 3000 7G is used to generate the resultant GeneChip array image.
Spotted Microarray and mirVana miRNA
Oligonucleotide Arrays: The human, mouse and rat 10K oligo sets obtained from MWG Biotech are received resuspended in 5x SSC at 40 μM concentration and are printed on epoxy coated slides (Corning) using the OmniGrid-100 at 60% humidity and room temperature. The slides are incubated overnight in a humid oven at 42oC. As these oligosets have 5’ aminolinkers there is no need for further processing.
mirVana miRNA Arrays: The mirVana miRNA probe set obtained from Ambion are received lyophilized and resuspended in 1x MWG Spotting Buffer at 50 μM concentration and printed on SCHOTT Nexterion Slide E microarray slides using the OmniGrid-100 at 60% humidity and room temperature. The slides are incubated overnight in a humid oven at 42oC.
Labeling of Nucleic Acids With Cy3 and Cy5 Fluorophores: We generate labeled nucleic acids for hybridization with a variety of commercially available kits. The primary consideration is that the labeling method employs an indirect labeling strategy rather than a direct one. Indirect labeling results in more efficient and equal labeling between the Cy3 and Cy5 probes than direct labeling does.
A standard amount of starting material for spotted microarray is 20 μg of total RNA per sample using the SuperScriptTM Indirect cDNA Labeling System. For samples with less than 20 μg of total RNA we use a method that employs a single round of amplification the Amino Allyl MessageAmpTM aRNA kit. For the mirVana miRNA arrays a standard amount of starting material is 20 μg using the mirVanaTM miRNA Labeling Kit.
Hybridization: The oligo arrays and the mirVana miRNA arrays are hybridized overnight at 42ºC with a commercially available buffer from Ambion.
Scanning: The laboratory uses an Axon 4000B scanner to generate the resultant DNA microarray images.
RNA Isolation
RNA for use in microarray experiments can be isolated in a number of ways. We have isolated RNA from both cell lines as well as tissue using TRIZOL per manufacturers suggestions and have had good results. One primary consideration, however, regardless of the procedure chosen for isolation, is that following isolation the RNA be checked for purity (A260/280 ratio of 1.8 or above), potential degradation and genomic DNA contamination. This can be achieved by spectrophotometry and gel electrophoresis.
Agilent Bioanalyzer
In our facility we employ the use of an Agilent Bioanalyzer which will assess the purity, potential degradation and genomic DNA contamination of RNA.
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