Branched rolling circle amplification method for measuring serum circulating micro<scp>RNA</scp> levels for early breast cancer detection

Abstract

Serum circulating microRNAs (c-miRNAs) are serving as useful biomarkers for cancer diagnosis. Here, we describe the development of a one-step branched rolling circle amplification (BRCA) method to measure serum c-miRNAs levels for early diagnosis of breast cancer. Four c-miRNAs, c-miRNA16 (c-miR-16), c-miRNA21 (c-miR-21), c-miRNA155 (c-miR-155), and c-miRNA195 (c-miR-195) were isolated from the serum of 49 breast cancer patients and 19 healthy controls. Among them, 45 breast cancer patients and 15 healthy controls were analyzed using one-step BRCA, 4 breast cancer patients and 4 healthy controls were analyzed by quantitative real-time PCR assay [corrected]. The serum levels of c-miR16, c-miR21, c-miR155, and c-miR195 were higher (P < 0.0001) in stage I breast cancer patients than healthy controls. These levels were also higher in several breast cancer molecular subtypes (HER-2 over-expression, Luminal A, Luminal B, and triple negative breast cancer) than in healthy control subjects. The diagnostic accuracy of c-miR16, c-miR21, c-miR155, and c-miR195 for early diagnosis of breast cancer was confirmed by receiver operating characteristic (ROC) curve assay. These results show that the BRCA method can be used to measure serum c-miRNAs levels, and that this method has high accuracy, sensitivity, and specificity. Moreover, both BRCA approach and quantitative real-time PCR (qRT-PCR) method show that the serum levels of c-miR16, c-miR21, c-miR155, and c-miR195 could be used as biomarkers to improve the early diagnosis of breast cancer, and distinguish different breast cancer molecular subtypes.