Título
RNA Purity, Real-Time PCR Sensitivity, and Colon Segment Influence mRNA Relative Expression in Murine Dextran Sodium Sulfate Experimental Colitis.
Autor
Bernardo Oldak
Mayra Yolanda Cruz Rivera
Ana Flisser Steinbruch
Fela Mendlovic Pasol
Nivel de Acceso
Acceso Abierto
Identificador alterno
doi: 10.7171/jbt.18-2903-001
Materias
Colitis experimental - ([Journal of Biomolecular Techniques (1524-0215) vol. 29(3), 61–70 (2018)]) Modelo DSS - ([Journal of Biomolecular Techniques (1524-0215) vol. 29(3), 61–70 (2018)]) IL-6 expression - ([Journal of Biomolecular Techniques (1524-0215) vol. 29(3), 61–70 (2018)]) Inflamación - ([Journal of Biomolecular Techniques (1524-0215) vol. 29(3), 61–70 (2018)]) BIOLOGÍA Y QUÍMICA - (CTI)
Resumen o descripción
The dextran sodium sulfate (DSS) model of colitis is widely used as a result of its simplicity and reproducibility and because it mimics clinicopathological disease features. Its effectiveness depends on the mouse strain, DSS MW, and brand. Quantitative RT-PCR (qRT-PCR) is highly sensitive for analyzing cytokine mRNA expression. We analyzed an acute model of DSS treatment in Balb/c mice for the onset of colitis using qRT-PCR for the quantification of a mouse cytokine transcript. We compared differences among 1--and 2-step qRT-PCR for transcript quantification, the effect of multiple concentrations of DSS, and the use of 2 reference genes in 3 portions of the colon. A reliable and sensitive 1-step protocol for qRT-PCR was established with a modified double LiCl precipitation for RNA isolation. The variability of 2 reference genes, ?-actin and eukaryotic elongation factor 2, was compared, and expression of IL-6 was analyzed in 3 segments of the colon. The RNA cleaning protocol prevented inhibition of qRT-PCR by DSS, and RNA loss was minimized. No clinical differences among the different DSS concentrations were seen on d 7, but higher concentrations resulted in the appearance of earlier symptoms. Higher efficiency and sensitivity of the 1-step qRT-PCR reaction using eukaryotic elongation factor 2 were obtained and also less variability. Although expression levels of IL-6 were high in the middle and distal colon, the middle section had consistently less variability in values. Thus, this segment is recommended for future studies. These factors influence the statistical significance of data and need to be considered to get accurate and reliable results and to improve comparisons of the published colitis experiments.
Editor
Association of Biomolecular Resource Facilities
Fecha de publicación
2018
Tipo de publicación
Artículo
Versión de la publicación
Versión publicada
Recurso de información
Formato
application/pdf
Fuente
Journal of Biomolecular Techniques (1524-0215) vol. 29(3), 61–70 (2018)
Idioma
Inglés
Relación
http://jbt.abrf.org/jbt-static/index.cfm/page/archives/jbt_archive.htm
Cobertura
US
Repositorio Orígen
Repositorio Institucional de la Facultad de Medicina,UNAM.Departamento de Microbiologia y Parasitologia
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