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.

Spotted Microarray

Oligonucleotide Arrays: The laboratory uses the mouse, and human OneArrayTM Microarray from Phalanx Biotech Group. These arrays contain approximately 26,000 and 30,000 mouse and human genome probes, respectively, with an additional 900-1000 experimental control probes per array.

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 1µg of total RNA per sample a method that employs a single round of amplification using the Amino Allyl MessageAmpTMII aRNA kit.

Hybridization: The oligo 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.