Protocols

Affymetrix GeneChip

Labeling of Target With Biotin: The laboratory uses a variety of commercially available assays for generating biotin-labeled samples for hybridization to Affymetrix GeneChips.

Hybridization: The GeneChip arrays are hybridized overnight in the Affymetrix hybridization oven at 42oC.

Washing and Staining: The Affymetrix GeneChip Fluidics Station 450 performs the automated Affymetrix wash and stain protocols.

Scanning: The Affymetrix GeneChip Scanner 3000 7G is used to generate the resultant GeneChip array image.

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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