High Resolution Melting (HRM) Optimization for Genotyping the SNP Rs3746444/Mir-499 in Vietnamese Breast Cancer Patients

Abstract

Single nucleotide polymorphism (SNPs) on microRNA region affects many biological processes by altering an interaction of miRNA and its targeted mRNAs. Due to potential role in cancer related-genes’ regulation and association with breast cancer implemented in many studies, the SNP rs3746444/miR-499 (A>G) can be suggested as a promising biomarker for early diagnosis of breast cancer. In this study, our aim was to develop an optimal High Resolution Melting (HRM) protocol for genotyping SNP rs3746444 in Vietnamese breast cancer patients. 100 patients were recruited in this experiment and DNA was extracted by modified-salting out method. HRM primers and further optimization were aided by in silico prediction, uMELT HETS. As a result, an optimal HRM condition was obtained with two control samples and used for genotyping. Then, allelic frequencies were calculated: f(A) = 0.76; f(G) = 0.19. Our frequencies showed a strong agreement with previous studies in Asia countries, which reported that allele G carrier may be a risk factor of breast cancer. Further association study between this SNP and breast cancer in Vietnam should be performed for more information about using this SNP as a biomarker to early diagnose for Vietnamese breast cancer. However, the optimal HRM condition may need more optimizing as our data is lacking of heterozygote control sample.