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. 2010 Feb 1;11:12. doi: 10.1186/1471-2199-11-12

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Copyright ©2010 Kheirelseid et al; licensee BioMed Central Ltd.

This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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Analysis of candidate EC genes using geNorm. (A): Average expression stability values of eligible EC genes. Expression stability of the control genes as calculated by geNorm. Stability value M is based on the average pair-wise variation between all genes. The least stable gene with highest M value was excluded and M value recalculated till end up with the most stable pair. (B): Determination of optimal number of control genes for normalisation. The GeNorm programme calculates a normalisation factor (NF) which is used to determine the optimal number of EC genes required for accurate normalisation. This factor is calculated using the variable V as the pairwise variation (Vn/Vn + 1) between two sequential NFs (NFn and NFn + 1). To meet the recommended cut off V-value which is the point at which it is unnecessary to include additional genes in a normalisation strategy. The recommended limit for V value is 0.15 but it is not always achievable. In this instance, the GeNorm output file indicated that the optimal number of genes required for normalisation was three.