Evaluation of the expression levels of BRAF mRNA in primary tumors of thyroid cancer using an ultrasensitive mutation assay.

The BRAF gene encodes for the mutant BRAF protein, which triggers downstream oncogenic signaling in thyroid cancer. Since most currently available methods have focused on detecting BRAF mutations in tumor DNA, there is limited information about the level of BRAF mRNA in primary tumors of thyroid cancer, and the diagnostic relevance of these RNA mutations is not known.Sixty-two patients with thyroid cancer and non-malignant thyroid disease were included in the study. Armed with an ultrasensitive technique for mRNA-based mutation analysis based on a two step RT-qPCR method, we analysed the expression levels of the mutated BRAF mRNA in formalin-fixed paraffin-embedded samples of thyroid tissues. Sanger sequencing for detection of BRAF DNA was performed in parallel for comparison and normalization of BRAF mRNA expression levels.The mRNA-based mutation detection assay enables detection of the BRAF mRNA transcripts in a 10,000-fold excess of wildtype BRAF counterparts. While BRAF mutations could be detected by Sanger sequencing in 13 out of 32 malignant thyroid cancer FFPE tissue samples, the mRNA-based assay detected mutations in additionally 5 cases, improving the detection rate from 40.6 to 56.3%. Furthermore, we observed a surprisingly large, 3-log variability, in the expression level of the BRAF mRNA in FFPE samples of thyroid cancer tissue.The expression levels of BRAF mRNA was characterized in the primary tumors of thyroid cancer using an ultrasensitive mRNA-based mutation assay. Our data inspires further studies on the prognostic and diagnostic relevance of the BRAF mRNA levels as a molecular biomarker for the diagnosis and monitoring of various genetic and malignant diseases.