Figure 1.
Validation of loading controls for Western blot throughout retinal development.
A representative Western blot of α-tubulin, β-actin and MAPK1 is shown in A for distinct retinal developmental stages (E18, P1, P4, P10, P14, P45). (B) Densitometric analysis of β-actin, α-tubulin and MAPK1. Results are presented as means ± SEM pooled from three independent experiments.
Figure 2.
Ct distribution of each putative reference gene among samples.
Cycle threshold (Ct) was determined for each reference gene tested and its distribution from 12 samples obtained from 6 distinct retinal developmental stages was determined (E18, P1, P4, P10, P14 and P45, 2 samples for each stage).
Figure 3.
Average expression stability calculated by stepwise exclusion with geNorm for the reference genes.
Average expression stability values were obtained for each experimental set (Groups 1–5). Pairwise variation decreases from left to right, due to stepwise exclusion of the least stable reference gene. M values below the theoretical threshold of 0.5 indicate adequate gene stability. The corresponding reference genes are ranked in Table 1. Group 1: E18, P1 and P4; Group 2: P1, P4 and P10; Group 3: P1, P4, P10, P14, P45; Group 4: P10, P14 and P45; Group 5: all ages.
Figure 4.
Pairwise variation analysis among sequential normalization factors calculated by geNorm.
Determination of the optimal number of control genes by calculation of the pairwise variation coefficient. A value from 0.15 to 0.20 was determined as an appropriate cut-off. To the right in each group, the increasing values are due to the inclusion of unstable reference genes. Group 1: E18, P1 and P4; Group 2: P1, P4 and P10; Group 3: P1, P4, P10, P14, P45; Group 4: P10, P14 and P45; Group 5: all ages.
Table 1.
Reference genes ranked by their expression stability (M) calculated by geNorm for different combination of samples.
Table 2.
Bestkeeper ranking for each group of samples with mean ± SD (standard deviation) and coefficient of variance (CV) of threshold cycle (Ct) values.
Table 3.
NormFinder analysis with reference genes ranked by their expression stability or different experimental sets.
Figure 5.
Effect of different choices of reference genes on the analysis of Cyclin D1.
Cyclin D1 mRNA content from samples of all retinal stages tested was normalized with Hprt1 and B2m (A), Mapk1 and Gapdh (B) Mapk1, Gapdh, Rn18s and Actb. The results of comparative Ct (ΔΔCt) method are represented as means ± SD for Group 5 (E18, P1, P4, P10, P14 and P45).
Table 4.
Adequate internal controls for different combinations of retinal developmental stages.