Scanning and Raw Data Analysis

The Bauer Core’s Axon scanners are the best choice for scanning most homemade arrays. We have two 4000B scanners which have a maximum resolution of 5µm. These scanners use dual lasers and a photo-multiplier tube (PMT) to excite and capture the fluorescent signal at wavelengths of 635 and 532 nm.

Axon scanner protocol (MS Word)

The GenePix software running the Axon scanners has a variety of features used to acquire the data from the scanned images. The GAL File (GenePix Array List) is used to find and identify each spot on the array, which allows the software to compute many parameters for each spot. GAL files are created using Excel sheets that identify the contents of each plate used to print the array. Training on GAL file creation costs can take from about half an hour to an hour.

Scanning Quality Control

There are several parameters one should check to determine the quality of each scanned array. This check can be performed either “on the fly”, by looking at several areas of the array after scanning, or by using the results table after analysis. There is also an Array Quality Control feature in the Reports section of GenePix which will look for the following criteria:

Signal to Background Ratio: An ideal array will have a signal to background ratio greater than 10. An array with a ratio greater than 5 is acceptable, but arrays with ratios of 3 or less may give poor-quality data. If the signal to background ratio is bad, consider optimizing the blocking procedure, including a blocking agent in the hybridization buffer, or optimizing the wash steps.
Signal to Noise Ratio: This value should be greater than 10.
Percentage of Features with Saturated Pixels: Try to keep this value below 0.1%.

Please direct questions about the scanners to Christian Daly (cdaly@cgr.harvard.edu).