Time-sequential change in gene expression after irradiation in glioblastoma (U373 MG) and lung cancer (A549)
The time-sequential change in immune-related gene expression of the glioblastoma cell line after irradiation was evaluated to speculate the effect of combined immunotherapy with radiotherapy. The same study was also performed using the A549 lung cancer cell line to compare the results. The human glioblastoma cell line, U373 MG (Uppsala), with inactive mutant p53, was used in this study. Cells were cultured in RPMI1640 media (Welgene), supplemented with 10% fetal bovine serum and 1% gentamicin, at 37 degrees Celsius in water saturated with 5% CO2. Ionizing irradiation (6 Gy) was delivered to the cell line (cell number, 5x10^5/100 mm dish) using 6 MV X-ray at 400 MU/min rate via a linear accelerator (Clinac 2100 C or Clinac 21EX, Varian Medical Systems). For comparison with a wild type cell line, a cell line of human lung cancer, A549, was also cultured under the same conditions.RNA isolation and Next-Generation Sequencing (NGS) were performed on the U373 MG and A549 cell lines before irradiation (0 hours, control group), and 6, 24, and 48 hours (test groups) post-irradiation. The total RNA concentration was calculated using the Quant-IT RiboGreen (Invitrogen). Samples were run on TapeStation RNA ScreenTape (Agilent) to assess the integrity of the total RNA. Only high-quality RNA preparations (RNA integrity number > 7.0) were used for RNA library construction. Indexed libraries were then sequenced on the HiSeq2500 platform (Illumina) by Macrogen Incorporated.A library was prepared using 1 microgram of total RNA from each sample using the Illumina TruSeq mRNA Sample Prep kit (Illumina, Inc.). First, the poly (A)-containing mRNA molecules were purified using poly-T-attached magnetic beads. Then, themRNA was fragmented into small pieces with divalent cations at elevated temperatures. SuperScript II reverse transcriptase (Invitrogen) and random primers were used to copy the cleaved RNA fragments into the first-strand cDNA. Subsequently, second-strand cDNA synthesis using DNA polymerase I and RNase H were performed. These cDNA fragments then underwent an end repair process, the addition of a single "A" base, and the ligation of indexing adapters. The products were purified and enriched viaPCR to generate the final cDNA library. The libraries were quantified using qPCR, according to the qPCR Quantification Protocol Guide (KAPA Library Quantification Kits for Illumina sequencing platforms), and validated using TapeStation D1000 ScreenTape (Agilent Technologies).