RNA Panel Sequencing Is an Effective Tool to Help Classify Splice Variants for Clinical Oncogenetic Diagnosis

Figure 2

Study of the PTEN c.206+6T>G variant. (a) RT-PCR analysis on blood sample RNA: peripheral blood of the patient with the PTEN c.206+6T>G variant was collected in PAXgene blood RNA tubes. RT-PCR analysis was performed with primers mapping to exons 2 and 5, and PCR products were separated by bioanalyzer electrophoresis. The 370 bp peak corresponds to the reference PTEN transcript, and the 325 bp peak corresponds to a PTEN transcript lacking exon 3. RT-PCR products were then analyzed by Sanger sequencing. (b) Minigene analysis: HeLa cells were transfected with pCAS2 vectors including wild-type or mutant PTEN sequences. Total RNA was isolated, RT-PCR analysis was performed using pCAS primers, and PCR products were separated by bioanalyzer electrophoresis. The 280 bp band corresponds to the reference PTEN transcript, and the 235 bp band corresponds to a PTEN transcript lacking exon 3. (c) PTEN immunohistochemistry: PTEN expression of the endometrium tumor was determined by immunohistochemistry on 3 μm paraffin sections with a PTEN rabbit antibody (Cell Signaling Technology). The PTEN IHC control slide was used to validate the technique.

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