Affymetrix Quality Control Services

All samples destined to be processed for hybridization to Affymetrix GeneChips must first be submitted to the MCF for sample quality control analysis (SAQC on MAID). Samples for expression analysis are analyzed for integrity, concentration and overall quality through use of the Agilent 2100 Bioanalyzer and the NanoDrop ND-1000 Spectrophotometer. Once a sample has passed QC it is then available for selection as an Affymetrix (AFFY) request in MAID.

Following processing of expression arrays and delivery of data to the user, the number one question to ask when looking at Affymetrix GeneChip® data is: "Was this a 'good' or a 'bad' chip?" There are several ways to do this, but the most important is to look at the QC file, a .PDF file provided by the MCF that is generated for every chip run in the core. The QC file is a summary of the chip, and provides important information about the quality of that chip. (QC on Affymetrix SNP Mapping Arrays is slightly different. If you have questions about SNP chips and QC, please contact the core).

Initially, the technician processing the sample conducts a visual inspection of the scanned image looking for any defects, areas of high background, or areas of low signal. As long as these areas do not represent more than 10% of the total probes for the chip, then the area can be masked and the data points thrown out as outliers without affecting the total quality of the data.

Affymetrix CEL File Image

The image to the right highlights some of the areas scrutinized by the MCF technician such as the spiked-in Oligo B2 control to check for hybridization uniformity. The border around the array, the corner region, the control regions in the center, are also checked to ensure the hybridization was successful.

Following visual inspection, the technician will examine the .RPT file (Report File) and check certain metrics for overall chip quality. The following are the most important factors to check in the RPT file*:

The Scaling Factor - In general, the scaling factor should be around three, but as long as it is not greater than five, the chip should be okay.
Percent Present Genes - This number can vary (especially depending on tissue type), but the % Present should be in the 40-50% range, as long as it is above 25%.
Sig (3'/5') - This is a ratio which that indicates how well the labeling reaction went. The two rations to really look at are your 3'/5' ratio for GAPDH and B-ACTIN. In general, they should be less than three. This, of course depends on labeling methodology.
Background (BG) - The average background signal generally should be less than 100.
Spike-In Controls (BioB, BioC, BioD, Cre) - These spike-in controls also tell how well your hybridization went. BioB is only present half of the time, but BioC, BioD, & Cre should always have a present (P) call.

More information/discussion about these metrics can be found in Nature Reviews Genetics: Expression Profiling - Best Practices for Data Generation and Interpretation in Clinical Trials. This paper gives a good introduction into the best practices for performing a microarray study.

*NOTE: These are general numbers to look for on an average array experiment using total RNA. They are not set in stone. If you are using aRNA, small-sample preparation, unique samples or tissues, etc. then these numbers will certainly be different. AS LONG THE METRICS ARE SIMILAR ACROSS ALL OF THE CHIPS IN A GIVEN EXPERIMENT, THEN THEY SHOULD BE "GOOD CHIPS" TO USE.

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