Background: A major challenge in the management of breast cancer (BC) is the search for biomarkers that are sensitive and less invasive. miRNAs are reported to be tissue-specific, stable and aberrantly expressed in different tumours, hence their emerging role as potential biomarkers in BC management.
Objective: The study focused on developing an in-house method for the detection of mir-145 and mir-21 using SYBR Green RT-qPCR in plasma samples of BC patients attending the Radiotherapy clinic.
Methods: Total RNA was extracted using glycogen modified TRIzol reagent and spin column from plasma samples. Extracted RNA was analyzed by NanoDrop (ND) spectrophotometry and confirmed using denaturing urea polyacrylamide gel electrophoresis (PAGE). Stem-loop reverse transcription (SLRT) primers for mir-145 and mir-21 were used to make cDNA. Real-time PCR amplification analysis was carried out in the presence of forward and universal reverse primers specific for mir-145and mir-21 conjugated using SYBR Green chemistry.
Results: ND Spectrophotometric analyses and smears of the PAGE image indicate the workability of the miRNA extraction method. Real time amplification curves showed that the miR-145 amplification and fluorescence correlated to an appreciable degree with the amount of RNA: S1, P4 (2.9 ng/l) > S7, P2 (5.4 ng/l) > S6, P3 (8.8 ng/l) > S2, P1 (4.3 ng/l) > S5, P6 (4.3 ng/l) > S3, P7 (3.2 ng/l) > S4, P8 (1.7 ng/l).
Conclusion: The RT-qPCR results validated the in-house technique for miRNA detection and showed the speed and sensitivity of the SL and SYBR Green I assay.
Key words: Breast cancer, miRNA, mir-145, mir-21, urea PAGE, SYBR Green RT-qPCR