Full Service Affymetrix Analysis of Client RNA Samples

I. Target Preparation

For eukaryotic samples, double-stranded cDNA is synthesized from total RNA or purified poly(A)+ messenger RNA isolated from tissue or cells. An in-vitro transcription (IVT) reaction is then done to produce biotin-labeled cRNA from the cDNA. The cRNA is fragmented before hybridization.

For E. coli samples, total RNA is isolated, followed by enrichment for messenger RNA. After fragmentation, the RNA is end-modified and conjugated with biotin.

II. Target Hybridization

A hybridization cocktail is prepared, including the fragmented target, probe array controls, BSA, and herring sperm DNA. It is then hybridized to the probe array during a 16-hour incubation. The hybridization process varies slightly for each of the different probe array types.

III. Experiment and Fluidics Station Setup

Specific experimental information is defined using Affymetrix® Microarray Suite Software. The probe array type, a sample description, and comments are entered and saved with a unique experiment name. The fluidics station is then prepared for use.

IV. Probe Array Washing and Staining

Immediately following hybridization, the probe array undergoes an automated washing and staining protocol on the fluidics station.

V. Probe Array Scan

Once the probe array has been hybridized, washed and stained, it is scanned. Each probe array is scanned twice, taking approximately ten minutes. The software calculates an average of the two images, defines the probe cells and computes an intensity for each cell. The double scan improves assay sensitivity and reduces background noise. Each complete probe array image is stored as a separate data file.

VI. Data Analysis

Data can be analyzed using a variety of software tools, such as the Affymetrix Microarray Suite 5.0 and the Affymetrix Data Mining Tools software package.

©2004 Harvard Medical School