Fig 1.
A: Electropherograms of RNA isolated from islets at different time of cultivation. RIN = RNA integrity number. B: RNA quantity per one islet and 28S:18S rRNA ratio in the same RNA samples.
Fig 2.
Effect of the length of cultivation on glucose-stimulated insulin secretion (GSIS) of pancreatic islets in vitro.
Glucose-stimulated insulin secretion was tested at three subsequent concentrations of glucose (3 mM, 22 mM, and 3 mM again). Data are given as a mean ± SD, n = 3.
Fig 3.
Dithizone staining of islets cultivated for different time periods.
Magnification 15×.
Fig 4.
Expression of candidate reference genes during cultivation.
Ct of individual candidate genes are shown as medians (lines), 25th percentile to the 75th percentile (boxes) and as ranges (whiskers) immediately after isolation and at 24, 48, 72, 96 and 120 hrs of cultivation. Data are based on at least 6 independent experiments. Statistical significance of the differences in Ct values was evaluated using Freidman’s Two Way ANOVA and multiple comparison method; *p < 0.05 48 vs. 0 hrs; # p < 0.05 72 vs. 0 hrs; †p < 0.05 96 vs. 0 hrs; ‡ p < 0.05 120 vs. 0 hrs; a p < 0.05 96 vs. 48 hrs; b p < 0.05 120 vs. 48 hrs.
Fig 5.
Expression of candidate reference genes during the first 24 hours of cultivation.
Ct of individual genes are shown as mean ± S.D., n = 6. Data are based on two independent experiments.
Fig 6.
Coefficient of variation (%) ΔCt(GOI-RG) at different time points of cultivation.
Data are based on at least 6 independent experiments.
Fig 7.
Expression stability values of candidate RGs at different phases of cultivation determined by GeNorm.
The M value represents an average stability measure of each possible combination of a particular RG with all other genes in the multiplex. The lower the M value of a given gene, the more consistent its expression relative to other genes in the multiplex.
Fig 8.
Expression stability of candidate RGs at different phases of cultivation evaluated according to the ±0.5 ΔCt rule.
Data are expressed as means ± SD, n = 6. Dashed lines indicate the interval of 0.7-1.4-fold change compared with the expression at 48 hrs.
Fig 9.
Relative and absolute quantification of F3, Gapdh and Ppia in isolated pancreatic islets during in vitro cultivation.
Relative quantification was calculated using the 2-ΔΔCt method related to time 48 hrs; absolute expression was determined using a calibration curve constructed for each specific transcript. RG pairs were chosen either by GeNorm, calculated for expression at 24 hrs only (Ppia/Ppib), or for the whole interval 0–120 hrs (Rplp2/Ppia) or by the ±0.5 ΔCt rule (Hprt/Ubc). When Ppia was evaluated as GOI, we used the next most stable reference gene identified by GeNorm (24 hrs: Ppib/Actb; 0–120 hrs: Rplp2/Ppib).