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969084.fig.003b
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Figure 3: Validation by PCR and Western blotting. (a): DCN mRNA expression in effusions and solid primary tumors, showing higher expression in the latter specimen type (P<.0001). DCN levels were measured by qRT-PCR using specific primers for decorin A1, A2 and B isoformes. The pool sample consisting of cDNA from five primary tumors and five effusion samples served as a positive control. DCN mRNA expression levels were normalized to RPLPO reference gene expression levels. (b): BCAR1 mRNA relative expression levels in primary tumors and effusions. Gene expression level was measured by semiquantitative RT-PCR and normalized to relative 28s expression. The MCF-7 breast carcinoma cell line served as positive control. BCAR1 levels were significantly higher in effusions (P<.0001). (c): VIL2 expression in effusions in comparison to primary carcinomas. VIL2 mRNA levels were measured by RT-PCR with specific primers and normalized to relative 28s expression. Human HT1080 fibrosarcoma cell line served as a positive control. VIL2 levels were significantly higher in effusions (P<.0001). (d): Total Ezrin protein expression in effusions in comparison to primary carcinomas. Ezrin protein levels were analyzed using specific antibodies and normalized to total beta-actin levels. Ezrin levels were significantly higher in effusions (P=.004).