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Prior to statistical analysis the data was normalised to the housekeeping genes glyceraldehyde 3-phosphate dehydrogenase (GAPDH), ribosomal phosphoprotein large P0 subunit (RPLP0) and transferrin receptor (TFRC). The normalisation factorwas calculated as the geometric mean of the three housekeeping genes. In addition a fourth root transformation of the data was conducted in order to reduce heteroscedasticity. In order to test differential expression at the different time points, the linear model; yijk=¦Ì+¦Ái+¦Âj+¦Åijk, was estimated for each gene separately on the normalised and transformed data. The y is the gene expression at time point i, in experiment j and parallel k. ¦Ì is the overall mean, ¦Ái is the effect of time point i, ¦Âj is the effect of experiment j and ¦Åijk is the random error between parallels from the same experiment (i=1,2,¡, 5, j=1,2,¡,4,and k=1,2,3). The experiment term is incorporated in order to correct for potential differences in maturity of isolated stromal¨Cvascular cells. In the following the estimated time effect is referred to as the estimated expression level.The fold change between two time points was calculated as the log2 of the ratio of the estimated expression levels for the two days, after transforming back to the original scale. For the proliferation phase the fold change was calculated between days 1 and 3, whereas for the differentiation phase fold changes were calculated between days 3 and 10. Genes were reported as significantly differentially expressed at different time points when the population marginal means were significantly different with level 0.05 according to the Tukey¨CKramer test for multiple comparisons. |
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