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Test array data analysis: Images will be analyzed by following quality control parameters as provided by Affymetrix:
a. 3'/5' ratio of housekeeping genes : This is a measure of the efficiency of the cDNA synthesis reaction. Reverse transcriptase synthesizes cDNA starting from the 3'-end of an mRNA and ending at the 5'-end. All Affymetrix arrays contain probes for the regions corresponding to 3', middle and 5'-end of the house keeping genes such as GAPDH and Actin. The ratio of signal intensity for 3' probes to that from 5' probes provides a measure of the number of cDNA synthesis reactions that went to completion (full length cDNA is synthesized). An ideal ratio would be 1 whereas a higher value indicates that many cDNAs were started but did not go to completion. The 3'/5' ratio for the housekeeping genes should be at most 3. If the ratio is above 3, some sensitivity of the assay may be lost.
b. Presence of spiked control cRNAs: Bio-B, C, D and CRE serve as a controls for hybridization and are spiked at the following concentrations: BioB: 1.5 pM, BioC: 5.0 pM, BioD: 25.0 pM, BioCRE: 100 pM. We specifically look at the average difference values which should be present in increasing amounts, B being the least and CRE the highest
c. Background values with standard deviation. Background value is a measure of the signal intensity caused by autofluorescence of the array surface as well as nonspecific binding of target or stain molecules (SAPE). The background values for all the arrays in one experiment should be very similar to each other; otherwise comparison data may not be accurate. Non-specific binding causes a low signal to noise ratio, which means that genes for transcripts present at very low levels in the sample may incorrectly be called absent. Thus, high background creates an overall loss of sensitivity in the experiment. |
d. Q value. Noise (Q value) results from small variations in the digitized signal observed by the scanner as it samples the probe array’s surface. It is measured by examining pixel-to-pixel variations in background intensities. The noise value for all the arrays in one experiment should be very similar to each other.
e. Scaling factor. The scaling factor provides a measure of the brightness of the array. The “brightness” (image intensity) varies from array to array. Non-biological factors (amount and quality of the cRNA, amount of stain or other experimental variation) can contribute to the overall variability in hybridization intensities. In order to reliably compare data from multiple arrays, it is essential that the intensity of the arrays be brought to the same level. Scaling is a mathematical technique used by the Microarray Suite Software (MAS) to minimize differences in overall signal intensities between two or more arrays thus allowing for more reliable detection of biologically relevant changes in the same sample. MAS calculates the overall intensity of an array by averaging the intensity values of every probe set on the array with the exception of the top and bottom 2% of the probe set intensities. The average intensity of the array is then multiplied by the Scaling factor to bring it to an arbitrary Target Intensity value (usually 1500) set by the user. Thus, scaling allows a number of experiments to become normalized to one Target Intensity, allowing comparison between any two experiments. In a particular set of experiment, the Scaling Factor value for all the arrays should be very close to each other (within three-fold of each other).
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