Coordinated regulation of gene expression by E ... - Jacobs University
Coordinated regulation of gene expression by E ... - Jacobs University
Coordinated regulation of gene expression by E ... - Jacobs University
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MATERIALS AND METHODS<br />
Figure 9: Overview <strong>of</strong> comparative <strong>gene</strong> <strong>expression</strong> <strong>of</strong> wild-type & fis (example) analysis using<br />
microarrays.<br />
2.2.13 Microarray analysis<br />
The scanned images <strong>of</strong> the spots on the arrays after quantification have to be subjected<br />
to normalization before any comparison <strong>of</strong> the <strong>gene</strong> <strong>expression</strong> is possible.<br />
Normalization <strong>of</strong> the spot intensity for a particular dye (Cy3 or Cy5) to the combined<br />
total intensity <strong>of</strong> that dye in all the spots <strong>of</strong> the array has to be performed for several<br />
reasons. It allows for comparison between microarray slides & between experiments<br />
and is necessary to eliminate the inherent difference in the intensities <strong>of</strong> Cy3 & Cy5<br />
fluorophores.<br />
Scanned array images were analyzed using the TM4 s<strong>of</strong>tware package from<br />
TIGR [Saeed et al., 2003]. The first step in the analysis, to quantify the spots and to do a<br />
quality check on the spots, was carried out using TIGR spotfinder s<strong>of</strong>tware. Briefly,<br />
spot intensities were quantified and quality <strong>of</strong> each spot was verified <strong>by</strong> calculating a<br />
quality control (QC) score depending on signal-to-noise ratio <strong>of</strong> both Cy3 and Cy5<br />
channels in the spot and shape quality <strong>of</strong> the spot. Then the p-values were calculated for<br />
each channel as result <strong>of</strong> a t-test comparing the spot pixel set and surrounding<br />
background pixel set still using the TIGR Spotfinder s<strong>of</strong>tware.<br />
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