title: Simultaneous separation of five major ribonucleic acids by capillary electrophoresis with laser-induced fluorescence in the presence of electroosmotic flow: application to the rapid screening of 5S rRNA from ovarian cancer cells.
abstract: RNA integrity is important in RNA studies because poor RNA quality may impact downstream methodologies. This study proposes a rapid and cost-effective method for the determination of RNA integrity based on CE-LIF in the presence of electroosmotic flow. The proposed method uses poly(ethylene) oxide (Mavg=4,000,000 Da) as a sieving matrix for total RNA separation. Ethidium bromide (μg mL(-1)) was dissolved in a polymer solution as an interchelating dye for on-column fluorescent labeling. The 28S rRNA, 18S rRNA, 5.8S rRNA, 5S rRNA and tRNA from the total human RNA extracted from the cells were fully separated using the proposed method. The lowest detectable concentration of total RNA achieved was 100 pg μL(-1) with a 6 min sample injection followed by on-column concentration. In addition, the temperature-induced degradation of total RNA was observed by CE-LIF. The electropherograms revealed more fragmentation of 28S and 18S rRNAs by temperature-induced hydrolysis compared with the 5.8S rRNA, 5S rRNA and tRNA. Therefore, the results indicated that RNA degradation should be considered for long-term, high-temperature incubations in RNA-related experiments involving RNA hybridization. The proposed method is furthermore, applied to the determination of 5S rRNA overexpressed in ovarian cancer cells as compared to the cervical cancer cells. Overall, CE-LIF is highly promising for rapid screening of ovarian cancers without tedious pre-amplification steps.
authors.affiliation: Department of Chemistry, Tunghai University, Taichung 40704, Taiwan.
authors.forename: Ya-Chu
authors.initials: YC
authors.lastname: Shih
authors.affiliation: Department of Chemistry, Tunghai University, Taichung 40704, Taiwan.
authors.forename: Ching-Ru
authors.initials: CR
authors.lastname: Liao
authors.affiliation: Molecular Medicine Research Center, Chang Gung University, Taoyuan 33302, Taiwan.
authors.forename: I-Che
authors.initials: IC
authors.lastname: Chung
authors.affiliation: Molecular Medicine Research Center, Chang Gung University, Taoyuan 33302, Taiwan.
authors.forename: Yu-Sun
authors.initials: YS
authors.lastname: Chang
authors.affiliation: Department of Chemistry, Tunghai University, Taichung 40704, Taiwan. Electronic address: poling@thu.edu.tw.
authors.forename: Po-Ling
authors.initials: PL
authors.lastname: Chang
mesh_terms: D045744:Cell Line, Tumor; D053841:Electroosmosis / Q000191:economics / Q000379:methods*; D019075:Electrophoresis, Capillary / Q000191:economics / Q000379:methods*; D004996:Ethidium / Q000737:chemistry; D005260:Female; D005453:Fluorescence; D006801:Humans; D007834:Lasers; D010051:Ovarian Neoplasms / Q000235:genetics*; D010053:Ovary / Q000378:metabolism; D012341:RNA, Ribosomal, 5S / Q000032:analysis* / Q000235:genetics; D015854:Up-Regulation
chemical_list: D012341:RNA, Ribosomal, 5S; D004996:Ethidium
keywords: Capillary electrophoresis; Ethidium bromide; Laser-induced fluorescence; On-column concentration; Ovarian cancer; Poly(ethylene oxide); Ribonucleic acid