The followings outline major steps of GeneChip expression analysis:
- Experimental Design and RNA Isolation: It is very important to carefully design your microarray experiments prior to isolation of RNA samples. For example, isolating RNA from a tissue with heterogeneous cell types may lead to results that reflect changes in the composition of different cell types rather than the changes of your interest. A proper selection of control RNA sample is also critical for interpretation of microarray data.
- 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 performed to produce biotin-labeled cRNA from the cDNA. Due to a short probe sequence (25-mer), the cRNA needs to be fragmented to around 100 ~ 200 bp long before hybridization. For prokaryotic samples, there are two protocols can be used: a) Total RNA followed by reverse transcription with random hexamers to produce cDNA. After fragmentation by DNase I, the cDNA is end-labeled with biotin by terminal transferase before hybridization, or b) Double-stranded cDNA is synthesized from poly-A RNA synthesized using Poly-Adenylated Protein (PAP). An in vitro transcription (IVT) reaction is then performed to produce biotin-labeled cRNA from the cDNA. The cRNA is fragmented before hybridization.
- Target Hybridization: A hybridization cocktail is prepared, including the fragmented target, probe array controls, BSA, and herring sperm DNA. The cocktail is then hybridized to the GeneChip for 16 hour.
- Fluidics Station Setup: Specific experimental information is defined using Affymetrix GeneChip Operating Software (GCOS) on a Microsoft Windows workstation. The GeneChip type, sample description, and comments are entered and saved with a unique experiment name. The fluidics station is then prepared for use by priming with the appropriate buffers.
- GeneChip Washing and Staining: Immediately following hybridization, the GeneChip undergoes an automated washing and staining protocol on the fluidics station controlled by GCOS software.
- GeneChip Scan: Once the probe array has been hybridized, washed, and stained, it is scanned. Each workstation running Affymetrix GCOS can control one scanner. The software defines the probe cells and computes intensity for each cell. Each complete GeneChip image is stored in a separate data file identified by the experiment name and is saved with a data image file (.dat) extension.
- Data Analysis: The .dat image is analyzed for probe intensities; many files will be generated, such as .CEL, .CHP, .EXP, .RPT and .TXT. Average intensity of each probe set for each GeneChip is reported in tabular formats and can be used for subsequent statistical analysis and data mining.
