ABSTRACT: To investigate the regulatory mechanisms governing the malignant signature of different gliomas we analyzed microRNA expression profiles in human tumor samples of world health organization (WHO) grade I (benign tumors), II (low grade tumors) and IV (high grade tumors) and from primary cultures obtained from tumor samples of grade II and IV. Patients This study included tumor samples histologically verified as astrocytic gliomas obtained from patients who had undergone craniotomy for microsurgical tumor removal. According to the revised WHO classification, tumors were diagnosed as: grade I or pilocytic astrocytomas; grade II or diffuse fibrillary astrocytomas; grade IV or glioblastoma multiforme. Primary cell cultures from grade II and grade IV gliomas were also obtained and miRNA expression in these cultures were analyzed RNA extraction Total RNA, including small RNA, was isolated from tissue samples using the mirVanaTM miRNA Isolation Kit (Ambion) following the standard protocol. The quantity and quality of the purified RNA was evaluated by spectrophotometric analysis and electrophoresis on denaturing gel of acrylamide. Multiplex Real-Time Quantitative Reverse-Transcriptase Polymerase Chain Reaction (RT-PCR) The miRNAs were first converted to cDNA using Multiplex RT for TaqMan Array Human MicroRNA Panel. The RT Master mix included 100 mM each of dNTPs , 50 U/ml MultiScrabe reverse transcriptase (Applied Biosystems), 20 U/µl RNase inhibitor (Applied Biosystems) and 10X RT Buffer. The 10 µl reactions, including 7 µl of RT master mix, 2 µl of purified microRNA and 1 µl of Multiplex RT Human primer pool (Applied Biosystem), were incubated in ice for 5 min and then in a thermal cycler for 30 min at 16°C, 30 min at 42°C, 5 min at 85°C, and then hold at 4°C. miRNA levels were normalized to the expression of small nucleolar RNAs, RNU44, RNU48 and RNU6B. All reverse transcriptase reactions, including no-template controls and RT controls, were run in duplicate. Real-time PCR was performed using a standard TaqMan PCR kit procedure on an “Real Time Fast 7900 HT” PCR System (Applied Biosystems). The 100 µl PCR included 50 µl RT product (before diluited 1:60) and 50 µl TaqMan Universal PCR Master Mix (2X) (Applied Biosystems). The total volume were loaded into Card TaqMan Low Density Array Human MicroRNA Panel (Applied Biosystem) including a total of 384 human microRNAs publicated on databases www.sanger.ac.uk. The reaction cards was runned at 50°C for 2 min and 95°C for 10 min, followed by 40 cycles of 97°C for 30s and 59,7°C for 1 min. All reactions were run in triplicate. Analysis of data was performed using the SDS 2.3 software using the 2-∆∆Ct (relative quantitative) method . The ∆Ct of every miRNA was determined in relation to the endogenous control RNA U6 that was invariably expressed in all samples. The ∆∆Ct value was determined in relation to the calibrator, namely the normal brain tissue. Resulting data were grouped according to the tumor grading e selectioned using a cut-off value of 3. Results were expressed as “fold change” over normal brain tissue. We analyzed two samples of grade I, two samples of grade II, two samples of grade IV gliomas. Four samples form norma brain were used as norma control. Primary cell cultures form grade II and grade IV samples were used for the analysis. All reverse transcriptase reactions, including no-template controls and RT controls, were run in triplicate.
