normalization data transformation protocol
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. ID_REF = VALUE = 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.
array scanning protocol
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.
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.
nucleic acid extraction protocol
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.