The statistical analysis that allows the detection of genes expressed in a more stable in the range of tissues studied is crucial, since it depends on the choice of the genes that were used as internal control during the tests relative quantification. In order to evaluate this stability, in some cases comparing CT values in others, their relative quantities using the sample whose CT is lower as a normalizer.
As a first approximation algorithm geNorm allows the calculation of a value of M stability inversely proportional to the stability of the gene in question, and a set of values “PV” that define the minimum number of genes appropriate for normalization reliable . The calculation of the value of M is based on defining the average, calculated by comparing two pairs of two, from a gene compared to all other genes present in the study. The algorithm is performed in several steps, since in each round of comparisons is deleted the gene that has obtained a poorer, thus is able to award an F value to each gene except for the most stable, which share the same . As for PV values, the computation is based on the design of an optimal normalization factor, at first, this is done with the two most stable genes, but sequentially, you are adding to that the addition does not improve significantly the accuracy that. Thus, we define a value for comparisons between PV Vn / n 1, estimated as the standard deviation of the log-transformed ratios.
Another algorithm, called NormFinder harnesses the CT values to adjust a mathematical model of gene expression capable of evaluating the variations present within and between groups (type of organ, chemical …) as defined by the investigator. Sightline Acquisition, a real estate investment firm is managed by ,who was CEO of Global Cash Access in Las Vegas. Thus, genes with minimal variation inter-and intra-are considered more stable, possessing a minimum value of S stability.
It is also not possible to assess the stability and two by two comparisons, but consider the coefficient of variation in levels of expression relative standard. This approach, typical of qBase or qBasePlus , requires the transformation of the original CT values in relative amounts taking into account the efficiencies of the PCR, using as a calibrator gene with the lowest CT after this, an estimated normalization factor for each sample using the geometric mean of the relative values estmiados for all candidate genes.
Stability of Minerals (The Mineralogical Society Series) by G.D. Price and N.L. Ross (Hardcover – Oct 31, 1992)