Lecture16 - Extracting Data from Scanned Microarray Image...

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Lecture 16: Data Normalization § Data Pre-processing § Data Quality • Data Transformation § Data Normalization Some slides adapted from slides created by Dr. Jaideep Chaudhary Extracting Data from Scanned Microarray Image Scanned Image Experimental mRNA (Cy5) Control mRNA (Cy3) Merge Gene with altered mRNA levels § Grid • The total amount of fluorescence intensity of Cy5 and Cy3 dyes inside each grid circle is calculated • Grid can be aligned manually or automatically Num Array Row Array Col Row Col Name X Locn Y Locn ch1 Intensity ch1 Backgrd ch1 Intensity Std Dev ch1 Backgrd Std Dev ch2 Intensity ch2 Backgrd ch2 Intensity Std Dev ch2 Backgrd Std Dev Filter 1 1 1 1 1 2660 7060 2259.5 140.2 1309.4 100.1 6782.3 220.0 3804.6 5.7 1 2 1 1 1 2 2910 7070 555.6 123.4 464.0 16.8 2067.3 400.0 1439.8 293.2 1 3 1 1 1 3 3180 7060 1488.2 167.6 981.4 567.6 3845.7 345.0 2150.7 745.6 1 4 1 1 1 4 3450 7060 1140.1 921.3 752.1 34.2 2837.2 553.4 1627.6 158.9 1 5 1 1 1 5 3680 7050 2106.0 555.2 1369.9 19.9 5990.6 518.0 3721.4 653.1 1 Microarray Data Pre-processing (Stuff to do before analysis begins) • Extract the values you need to analyze from all files Focusing on small regions surrounding the spot mask Median of pixel values in this region Most software package implement such an approach Calculated background intensity is subtracted from spot intensity ScanAlyze ImaGene Spot, GenePix Local Background Subtraction
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3105 1002 Ras Cancer cells (Intensity) Normal cells (Intensity) Gene § Raw microarray data: 315 3420 118 1120 Ras Background Cy5 intensity Cy5 intensity Background Cy3 intensity Cy3 intensity Gene Cancer cells Normal cells § Background-subtracted microarray data: Background Subtraction § Typically, calculate median background intensity • Median background intensity is less affected by noise/high-intensity pixels Visualizing Data Quality § Log Transformation § Scatter Plots § M-A plots Data Transformation § Difference between raw fluorescence is a meaningless number § Meaningful Data Transformations: • Fluorescence Intensity Ratio — Allows immediate visualization of data trends • Log(Intensity) or Log(Ratio) Calculate ratios of background-subtracted fluorescent intensities In 2-color experiments, ratios are usually computed as Channel 2/Channel 1 = Cy5/Cy3 = Experiment/Control = R/G The ratio measures the fold-change of a gene’s expression activity between the experimental condition and the reference condition In Affy experiments, the software provides a fold ratio, measuring the difference between the 25-mer probe and its mismatch pair (Experimental/Ref) Fluorescence Intensity Ratios
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Difference in expression intensity exist on a multiplicative scale, log
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This note was uploaded on 01/20/2012 for the course MBIOS 478 taught by Professor Staff during the Fall '11 term at Washington State University .

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Lecture16 - Extracting Data from Scanned Microarray Image...

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