Table 1.
Endogenous control genes analysed with geNorm software in this study.
Table 2.
Cp values of endogenous control genes in ocular surface epithelial regions.
Table 3.
Normalisation factor calculated with stable pair of endogenous control genes.
Table 4.
Ranking of the endogenous control genes with increasing stability in descending order in ocular surface regions.
Figure 1.
Distribution of cp values of ECGs in different OS epithelial regions.
Figure 1A, the scatter graph of the Cp values of all the ECGs shows all the ECGs had cp value≤35. The order of samples 1–12 were as follows: Samples 1, 2, 3 were cornea 1, cornea 2, cornea 3, samples 4, 5, 6 were limbus 1, limbus 2, limbus 3, samples 7, 8, 9 were LEC 1, LEC 2, LEC 3 and samples 10, 11, 12 were conjunctiva 1, conjunctiva 2, conjunctiva 3. Figure 1B is a box plot of q PCR cp values of the ECGs. Each box plot is mean Cp value of all OS epithelial regions. The median Cp value is represented as black line within the box plot, and it divides the Cp values into lower and upper quartile ranges. The whiskers represent the upper and lower data range for the samples tested. The box plot of PPIA demonstrates tight distribution of the samples around the median value. Similarly box plot of RPLP0 shows uniform distribution of Cp values around the median with tight whisker distribution.
Figure 2.
Average expression stability values of endogenous control genes in ocular surface regions.
The M values on the Y axis measures gene expression stability. Higher the M value the lower is the gene stability and vice versa. In graphs A, B, C, D, E the gene stability is represented in increasing order of stability from left to right. In graphs F, G, H, I, J, pair wise variation is represented. The pair wise variation for V2/3 is less than 0.15 for all the regions represented by graphs F, G, H, I and J. Hence Normalisation Factor (NF) calculated with these two genes is stable and would not significantly change with addition of a third endogenous control gene.
Figure 3.
Normalised gene expression of Dopachrome Tautomerase (DCT) in ocular surface regions.
Graph A shows the normalised expression of Dopachrome Tautomerase (DCT) in OS epithelial regions using the 10 individual ECGs studied. As seen in graph A, DCT was found to have highly variable expression with individual endogenous control genes. Graph B shows normalised expression of DCT using the normalisation factor (NF) as determined in this study. Normalised expression of DCT with NF is stable and some expression of DCT is also noted in limbus.