Figure 2. Gene expression analysis by qPCR.
(a) Hierarchical clustering analysis and differential expression of 18 genes from SVir library. Hemocyte gene expression profiles were analysed in biological replicates from oysters: non injected as control (C), surviving infections with avirulent V. tasmaniensis LMG 20012T (T), with virulent V. splendidus LGP32 (S) and virulent V. aestuarianus LPi 02/41 (A). Each biological replicata was constituted by a pool of hemocyte RNA from ten oysters, and between 2 and 5 replicates were analysed for each experimental condition. Each cell in the matrix corresponds to the expression level of one gene in a sample. The intensity of the color from green to red indicates the magnitude of differential expression (see color scale at the bottom of the image). Relative expressions were calculated according the 2−(ΔΔCt) method normalized with elongation factor-1α (EF-1α). Each value was calculated in reference to the mean of ΔCt of all conditions (relative expression = 1). The dendrograms at the top of the figures indicate relationship among experimental conditions which define clusters of conditions (CC). The dendrograms at the left of the figures indicate relationship among the profiles of the selected genes which define clusters of expression (CE), after clustering analysis using Multiple Array Viewer software. Gene #1: Inhibitor of apoptosis; #2: Baculoviral IAP repeat-containing protein 4; #3: Enhancer of kinase suppressor of Ras2; #4: Rac GTPase-activating protein1; #5: Proteasome 216S subunit, non-ATPase 11a; #6: Glyceraldhyde 3-phosphate dehydrogenase; #7: Thioredoxin; #8: C-type lectin 2 like protein; #9: Metallothionein IV; #10: F-box only protein37; #11: Cystatin B-like protein; #12: Heat shock protein 22 isoform 1; #13: L-rhamnose-binding lectin; #14: Microsomal glutathione S-tranferase; #15: Cystatin A; #16: Interferon-induced protein 44; #17: Cullin-associated and neddylation-dissociated 1. Hierarchical clustering was contructed with Multiple Array Viewer software using average linkage clustering with Pearson correlation as the default distance metric. (b) Examples of gene expression profiles defining pathogen- or challenge-specific signatures. SVir genes; line 1: similar response to both virulent strains; line 2: response induced by V. splendidus LGP32; line 3: response induced by V. aestuarianus LPi 02/41; line 4: SaVir genes. Different letters indicate significant variation between conditions (p<0.05) determined using the non-parametric multiple comparison test ANOVA of Kruskal-Wallis.