Joachim Li:Microarray Scanning
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GenePix 4000a Microarray Scanner Protocol Unknown Author, Joachim Li Lab - Unknown Date
2) Slide scanner door open. Insert chip hyb side down with label facing towards you into scanner. Clip the chip holder easily around the slide. DO NOT PUSH DIRECTLY DOWN ON THE CLIP! 3) Set PMT's to 600 in both 635nm(Cy5) and 532nm(Cy3) channels. These settings are controlled in the "HARDWARE SETTINGS" window. 4) Perform low resolution "PREVIEW SCAN" to determine location of spots and initial hyb intensities. 5) Once you have determined scan location, draw a "SCAN AREA" border around the entire array. 6) Perform a quick visual inspection of hyb and make initial adjustments to PMT's. 7) For gene-expression hybs, you would like the ratio over the entire scan area to be 1.0. You will want to raise or lower the red and green PMT's to achieve this color balance. This will be performed more stringently later. 8) Before you perform your data scan, change the "LINES TO AVERAGE" in the "HARDWARE SETTINGS" to 2. The scanner will now scan each pixel twice and average the data counts collected which reduces any background noise that may be present. 9) Perform a high-resolution "DATA-SCAN". As the image is scanning, go to the "HISTOGRAM" tab located at the top of the screen. The histogram allows you to dynamically observe the relative intensities of both channels as you scan. Important components of the histogram tab are: - Histogram settings should be: Image: Both X-axis: Fullscale Y-axis: Log Axis on Fullscale - The histogram is basically showing you the percentage of Normalized Counts that are at a given intensity or quanta. - The hisotgram only shows you the pixels you are viewing in the image tab, i.e., if you are zoomed in on the image it will only show you the pixels you are zoomed in on. - Remember that EVERY pixel is represented in the histogram, so artifacts and dirt will skew the readings. If you have a lot of dirt or artifacts, try and zoom in on a clean portion of the array to determine accurate PMT settings. 10) Observe the histograms and make adjustments to the PMT's. In generla, you would like to see pixels represented across the entire intensity range. However, saturated pixels (with counts greater than ~67,000) will be thrown out and spots with pixel counts close to background will result in poor data. 11) Once the PMT levels have been set so that the intensity ratio is near 1.00 perform a "DATA SCAN" over the "SCAN AREA" and save the results. 12) To save your image, go to the "OPEN/SAVE" button and select "SAVE IMAGES". 13) We like to save as type=Multi-image TIFF files and using a naming convention with includes a date prefix. In general, it is not necessary to save the Preview or Export images, and instead only save the 635nm and 532nm wavelengths. 14) Transfer your data to CD media or a USB drive. |
