RNA disruption is associated with response to multiple classes of chemotherapy drugs in tumor cell lines

Narendrula, Rashmi; Mispel-Beyer, Kyle; Guo, Baoqing; Parissenti, Amadeo M.; Pritzker, Laura B.; Pritzker, Ken; Masilamani, Twinkle; Wang, Xiaohui; Lannér, Carita

Total RNA was isolated from A2780 cells treated with or without 0.2 μM docetaxel for 48 h. The component RNAs (5 μg per lane) were resolved by denaturing formaldehyde 1 % agarose gel electrophoresis, transferred to BioTrace PVDF membranes (Life Sciences, Pensacola, FL, USA), and UV cross-linked. The membranes were then incubated with various radiolabeled probes hybridizing to sequences within the 28S and 18S rRNAs. These probes ( Table 1) were derived from rRNA probes described in publications by He et al. [24] , Houge et al. [12] and Nadano et al. [25] . The alignment of all probe sequences were checked against human rRNA sequences (28S rRNA: Genbank ID M11167.1; 18S rRNA: Genbank ID M10098.1) to ensure complete sequence homology. Probes were labeled using γ-32 P-ATP and the DNA 5' End Labeling System by Promega (Fisher Scientific, Mississauga, ON, CA). Hybridization was performed according to Brown and Mackey [26] . Following hybridization and washing, blots were sealed in bags and exposed to phosphor imaging screens for various lengths of time. Screens were scanned using a Bio-Rad Molecular Imager FX (Bio-Rad Laboratories, Ltd., Mississauga, ON, CA). Band sizes were determined using Quantity One software from Bio-Rad Laboratories, Inc.