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

Figure 3

Study of the MLH1 c.791-489_791-20del variant. (a) Pedigree: fill symbols indicate patients affected with cancer. Open symbols indicated relatives unaffected with cancer. The type of cancer and age at presentation are given under the symbol. Red dots indicate carriers of the MLH1 variant; green dots indicate people who do not carry the MLH1 variant. (b) RT-PCR analysis on blood RNA: peripheral blood of the patient with the MLH1 c.791-489_791-20del variant was collected in PAXgene blood RNA tubes. RT-PCR analysis was performed with primers forward and reverse mapping to exons 8 and 11, respectively, and PCR products were separated by bioanalyzer electrophoresis. RT-PCR products were then analyzed by Sanger sequencing. (c) Minigene analysis: HeLa cells were transfected with wild-type or mutant plasmids. Total RNA was isolated, RT-PCR analysis was performed, and PCR products were separated by bioanalyzer electrophoresis. The 338 bp band corresponds to the reference MLH1 transcript, and the 244-bp band corresponds to a MLH1 transcript lacking exon 10.

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