array scanning protocol
normalization data transformation protocol
The normalization and all the data analysis were performed according to the manufacturers instructions using their web-based software package: http://sabiosciences.com/pcrarraydataanalysis.php The Arrays contained specific primers for 352 known human microRNA, 4 housekeeping genes (HKGs; hsa-SNORD48, hsa-SNORD47, hsa-SNORD44, and hsa-U6), 2 RNA-quality, and 2 polymerase chain reaction (PCR)–positive controls. The ΔΔ Ct method was used to calculate the relative expression of microRNA in the control and astrocytic tumors. MicroRNAs with fold changes of 2 and with significant p value (p≤ 0.05) between the two groups were considered to be differentially expressed. ID_REF = VALUE = normalized signal (against housekeeping genes)
For Real-Time PCR, the appropriated RT2 SYBR Green qPCR Master Mixes (SABiosciences) was added to the mix containing the first strand diluted and RNAse-free water.
nucleic acid extraction protocol
Total RNA isolation (30mg) was performed using the TRIzol® protocol (Invitrogen, USA) according to the manufacturer′s instructions