Advantages of accurate normalization

What is the advantage of additional ICs? One may consider that the variability between the expression of any two samples is due to three factors: (1) true differences in expression, (2) differences in the RNA content of the sample,

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18SrRNA ARP

Internal control b-actin

Figure 6.4

Comparison of expression levels of three internal control (ICs) genes across in 5 samples of murine retina. ICs used are: 18S rRNA, acidic ribosomal phosphoprotein (ARP) and p-actin. A. IC expression corrected to the mean expression of that IC across all five samples, illustrating similar profiles of expression for all three genes. The geometric mean of all three genes is also shown. B. The variance within each sample can be measured, and the contribution to the total sum of squares (SStota) determined. Sample 1 demonstrates the greatest contribution to the contribution to the SStotai, whereas Sample 3 demonstrates the highest correlation between expression values. C. Contribution to SStota/ by gene, showing that 18S rRNA demonstrates a greater variance than either ARP or p-actin.

and (3) measurement errors. True differences in expression will reflect basal expression levels as well as changes in response to stimuli, and will include biological variation (6.1.2). It is this level of expression that is the desired endpoint of any experiment. Measurement errors are present in any assay, and should be minimal with most qPCR platforms when analyzing samples in parallel (ideally well below 10%). Differences in RNA load can have a dramatic effect upon the normalized expression. Poor normalization can produce quite misleading data, and for this reason the use of at least two ICs is recommended to ensure that the results obtained are not simply an artifact of normalization. Use of additional ICs also will provide a more accurate measure of RNA loading, and this should ensure that this source of error is minimized. The end result should be a reduction in the overall noise, enabling an improved assay resolution and a better ability to discriminate differences. Or in simple terms, more ICs should result in smaller error bars.

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