4 - Central Institute of Brackishwater Aquaculture
4 - Central Institute of Brackishwater Aquaculture
4 - Central Institute of Brackishwater Aquaculture
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Natlonal Workshop-cum-Trainiw on Bioinformat4u and InfOrtnati~n Management in <strong>Aquaculture</strong><br />
Cy5. The amount <strong>of</strong> signal emitted is directly in proportion to the amount <strong>of</strong> dye<br />
at the spot on the microarray and these values are obtained and quatitated on<br />
the scanner. This results in the image that is typical microarray picture.<br />
3.2. Preprocessing<br />
Preprocessing <strong>of</strong> microarray data comprises analytical or transformational<br />
procedures that is directed at resolving the systematic error and bias.<br />
3.2.1. Checking the Background Information: Background and foreground<br />
intensities together form the spot intensity. If the spot intensities are dependent<br />
on the background intensities, it is possible either not to apply any background<br />
correction to the data or discard the deviating observations from further<br />
analyses. The background corrected intensity values are calculated spotwise by<br />
subtracting the background intensity from the spot (foreground) intensity.<br />
3.2.2. Calculation <strong>of</strong> Expression Change: There are three commonly used<br />
measures <strong>of</strong> expression change. Intensity ratio is the raw expression value, and<br />
log ratio and fold change are transformationally derived from it.<br />
(i) Intensity Ratio: After background correction, expression change is<br />
calculated. The simplest approach<br />
is to divide the intensity <strong>of</strong> a gene in the sample by the intensity level <strong>of</strong> the<br />
same gene in the control. Intensity ratio can be calculated from background<br />
corrected<br />
or uncorrected data (here we use corrected data) The formula for two-color<br />
data is<br />
Cy3'<br />
Intmliy rauo = -<br />
C 5'<br />
For affymetrix data, substitute Cy3' and Cy5' with the appropriate intensitiesfrom<br />
the sample and control chips. This intensity ratio is one for an unchanged<br />
expression, less than one for down regulated genes and larger than one for upregulated<br />
genes. The problem with intensity ratio is that its distribution is highly<br />
asymmetric or skewed. Up-regulated genes can take any values from one to<br />
infinity (1 to X) whereas all down regulated genes are squeezed between zero<br />
and one (0 to 1). Such distributions are not very useful for statistical testing.<br />
(ii) Log Ratio: To make the distribution more symmetric (normal-like) logtransformation<br />
can be applied. The most commonly used log-transformation is 2-<br />
based (log 2), but it does not matter whether you use natural logarithm (loge) or<br />
base 10 logarithm (log10) as long as it is applied consistently to all the samples.<br />
The formula for the calculation <strong>of</strong> 1092-transformation is:<br />
Lng ratto = log:(Intemtt). ratlo) = log:!<br />
(:;,;:) -<br />
After the log-transformation, unchanged expression is zero, and both<br />
upregulated and down-regulated genes can take values from zero to infinity. Log<br />
ratio has some nice properties compared with other measures <strong>of</strong> expression. It<br />
makes skewed distributions more symmetrical, so that the picture <strong>of</strong> variation<br />
becomes more realistic. In addition, normalization procedures become additive.
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