Balkan Journal of Medical Genetics

MicroRNAs IN BREAST CANCER —OUR INITIAL RESULTS
Popovska-Jankovic K1, Noveski P1, Chakalova L1, Petrusevska G2, Kubelka K3, Plaseska-Karanfilska D1
*Corresponding Author: Professor Dr. Dijana Plaseska-Karanfilska, Research Centre for Genetic Engineering and Biotechnology “Georgi D. Efremov,” Macedonian Academy of Sciences and Arts, Krste Misirkov 2, Skopje 1000, Republic of Macedonia; Tel: +389(0)2 3235410; Fax: +389 (0)2 3115434; E-mail: dijana@manu.edu.mk
page: 87

MATERIALS AND METHODS

Fresh frozen tissues (normal and malignant) from patients with breast cancer were obtained from the Institute of Pathology, Medical Faculty, Skopje, Republic of Macedonia. Histopathological data were obtained from all patients. The protocol used for miRNA quantification in tissue samples included three separate procedures: extraction of total RNA, RT- and ReTi-PCR assays. RNA extraction was performed using an RNeasy Kit (Qiagen). Quality and quantity of total RNAs were checked on 1.0% agarose gels and a nanodrop spectrophotometer. RNA samples were dissolved in RNase-free water and stored at –80°C. MicroRNA quantitation was performed by stem-loop RT-PCR followed by Taq-Man® PCR analysis [13] using TaqMan® MicroRNA Reverse Transcription Kit, TaqMan® Universal PCR Master Mix and five TaqMan® MicroRNA Assays (miR-155, miR21, miR-125b and miR-145 and RNU6b as a control gene) (Life Technologies). The RT-PCR mix was made on ice in a final volume of 15 mL, following the manufacturer’s protocol. Thermal cycling conditions were: 30 min. at 16°C, 30 min. at 42°C and 5 min. at 85°C. The ReTi-PCR assay was performed in duplicates in a total volume of 20 mL consisting of 7.7 mL ddH2O, 1.0 mL 20 ´ TaqMan® Small RNA Assay, 10.0 mL 2 ´ TaqMan® Universal PCR Master Mix and 1.3 mL RT reaction product (Life Technologies). Thermal cycling conditions were: enzyme activation and initial denaturation at 95°C for 10 min., followed by 40 cycles of 15 seconds at 95°C and 60 seconds at 60°C. The expression of each miRNA relative to RNU6b was determined using the DDCt method. MicroRNA microarray analysis was done using a complete labeling and hybridization kit (Agilent Technologies, Santa Clara, CA, USA) following the manufacturer’s protocol [14].

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