Mirneasy ffpe minikit

Total RNA to quantitate both MGMT mRNA and miRNAs was extracted from formalin-free alcoholic-based fixative FineFIX^® and paraffin embedded samples by MiRNeasy FFPE minikit (Qiagen, Venlo, Netherlands). Reference RNA from healthy brain was purchased from Clontech (Clontech Laboratories, Mountain View, CA, USA) and obtained from the whole brain of a 28-yr-old Asian male deceased because of sudden death. MicroRNA expression in glioblastoma and healthy brain RNA samples was firstly calculated relative to U6 snRNA. Samples from glioblastoma were subsequently expressed as Fold Changes (FC) in respect to reference RNA from healthy brain tissue. Total RNA from T98G cells was isolated using Tri-reagent^™ (Sigma Aldrich). The 2100 bioanalyzer was used to determine the integrity and measure the concentration of total RNA samples (Agilent Technologies, Instrument DE54700480, Eukaryote Total RNA Nano Series II.xsy).

RNA to quantitate both IL-8 mRNA, VEGF mRNAs and miR-93 was extracted from formalin-free alcoholic-based fixative FineFIX and paraffin embedded samples of the archive of deceased patients by MiRNeasy FFPE minikit (Qiagen, Venlo, Limburg, Netherlands). Reference RNA from healthy brain was purchased from Clontech (Clontech Laboratories, Mountain View, CA, USA) and obtained from the whole brain of a 28-yr-old Asian male deceased because of sudden death. Mir-93 expression in LGG, HGG and healthy brain RNA samples was firstly calculated relative to U6 snRNA. Samples from LGG and HGG were subsequently expressed as Fold Changes (FC) in respect to Clontech reference RNA obtained from healthy brain tissue. Total RNA from U251 cells and T98G cells was isolated using Tri-reagent (Sigma Aldrich). The 2100 bioanalyzer was used to determine the integrity and measure the concentration of total RNA samples (Agilent Technologies, Instrument DE54700480, Eukaryote Total RNA Nano Series II.xsy).