GBM cell culture in PLA under 2D vs 3D for day 4,7,14
At Day 4, 7, 14, total RNA of GBM cells was isolated with Trizol followed by manufacturer’s instruction (Invitrogen, San Diego, CA). For 2D cell cultures, regular isolation protocol was adopted. For 3D cell cultures, the scaffolds contain cells were cut into small pieces by scissors, then 1mL Trizol was added to the mixture. Total RNA was purified by Qigen RNeasy mini kit (Qiagen, Velencia, CA). Purified RNA was analyzed on a ND-1000 spectrophotometer (Nanodrop Technologies, Wilmington, DE). The quality and quantity of total RNA were verified using a 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA). Three independent RNA samples at each time point with 260/280 ratios between 1.9 and 2.0 and the RIN(RNA integrity number) was greater than 8.5 were pooled for subsequent analysis. Single and double stranded cDNA was synthesized from total RNA samples using SuperScript II (Invitrogen, CA, USA). High quality total RNA (250 ng) was used as the starting material. The genechip 3' IVT expression kit was used for the first-strand, second-strand cDNA Synthesis, and in vitro transcription to synthesize labeled cRNA. The cRNA was then purified and fragmented for hybridization analysis. 12.5 mg aliquants of the fragmented cRNA were hybridized with the Primeview array (Affymetrix, Santa Clara, CA) in hybridization cocktail (0.5 mg/ml cRNA, 50 pM control oligonucleotide B2, 1.5 pM bioB, 5 pM bioC, 25 pM bioD, 100 pM cre, 0.1 mg/ml herring sperm DNA, 0.5 mg/ml acetylated BSA, 100mM MES, 1 M Na+, 20 mM EDTA, 0.01% Tween 20, 10%DMSO). Hybridization was allowed to proceed overnight (16 h) at 45°C, 60 rpm, followed by washing and staining with the Affymetrix hybridization kit (Affymetrix, Santa Clara, CA). Hybridization assay procedures including preparation of solutions were carried out as described in the Affymetrix GeneChip Expression Analysis Technical Manual. The distribution of fluorescent material on the array was obtained using 7G3000 GeneChip Scanner (Affymetrix, Santa Clara, CA). Microarray Suite (MAS) version 5.0 and Affymetrix Genechip Command Console (AGCC) supplied by Affymetrix was used for gene expression analysis.