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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.
Shih, Ya-Chu; Liao, Ching-Ru; Chung, I-Che; Chang, Yu-Sun; Chang, Po-Ling.
Afiliación
  • Shih YC; Department of Chemistry, Tunghai University, Taichung 40704, Taiwan.
  • Liao CR; Department of Chemistry, Tunghai University, Taichung 40704, Taiwan.
  • Chung IC; Molecular Medicine Research Center, Chang Gung University, Taoyuan 33302, Taiwan.
  • Chang YS; Molecular Medicine Research Center, Chang Gung University, Taoyuan 33302, Taiwan.
  • Chang PL; Department of Chemistry, Tunghai University, Taichung 40704, Taiwan. Electronic address: poling@thu.edu.tw.
Anal Chim Acta ; 847: 73-9, 2014 Oct 17.
Article en En | MEDLINE | ID: mdl-25261903
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.
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Texto completo: 1 Colección: 01-internacional Base de datos: MEDLINE Asunto principal: Neoplasias Ováricas / ARN Ribosómico 5S / Electroforesis Capilar / Electroósmosis Tipo de estudio: Diagnostic_studies / Screening_studies Límite: Female / Humans Idioma: En Revista: Anal Chim Acta Año: 2014 Tipo del documento: Article País de afiliación: Taiwán Pais de publicación: Países Bajos
Texto completo
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Texto completo: 1 Colección: 01-internacional Base de datos: MEDLINE Asunto principal: Neoplasias Ováricas / ARN Ribosómico 5S / Electroforesis Capilar / Electroósmosis Tipo de estudio: Diagnostic_studies / Screening_studies Límite: Female / Humans Idioma: En Revista: Anal Chim Acta Año: 2014 Tipo del documento: Article País de afiliación: Taiwán Pais de publicación: Países Bajos