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Materials and Methods Figs. S1 to S13 References Other Supporting Online Material for this manuscript includes the following: Tables S1 to S3Supplementary Materials and Methods

By Yiping He, Bert Vogelstein, Victor E. Velculescu, Nickolas Papadopoulos, Kenneth W. Kinzler and Rna Preparation

Abstract

DNA-free total RNA from Jurkat T cell-leukemia line and MRC5 diploid lung cell line were purchased from Ambion (Austin, Tx). The colorectal cancer cell line HCT116 and pancreatic cancer cell line MiaPaCa2 were purchased from ATCC (Manassas, VA) and were grown in McCoy's 5A medium with 10 % fetal calf serum. Normal human peripheral blood mononuclear cells (PBMC) from a healthy volunteer were isolated from fresh peripheral blood with Histopaque-1077 (Sigma, St. Louis, MO). Total RNA from PBMC, HCT116 and MiaPaCa2 was isolated with the RNeasy kit (Qiagen, Valencia, CA) according to the manufacturer’s instructions. The DNA was removed from the total RNA preparation with the DNA-free kit (Ambion). Total RNA was enriched for the non-ribosomal fraction by treating 20-24 ug of DNA-free total RNA with the RiboMinus transcriptome isolation Kit (Invitrogen, Carlsbad, CA). To remove as much ribosomal RNA (rRNA) as possible, two rounds of rRNA reduction were performed. Subsequently, the RNA was ethanol-precipitated, washed in 70 % ethanol, resuspended in RNase-free water, and finally eluted in RNase-free water from a RNeasy column. To confirm that each RNA sample was DNA-free and to evaluate the effectivenes

Year: 2008
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