期刊
ANALYTICAL BIOCHEMISTRY
卷 413, 期 1, 页码 24-29出版社
ACADEMIC PRESS INC ELSEVIER SCIENCE
DOI: 10.1016/j.ab.2011.01.037
关键词
beta-Actin; GAPDH; Internal control; Monocyte; Neonate; Western blot
资金
- National Science Council, Taiwan [NSC 98-2314-B-182A-004-MY3]
- Chang Gung Memorial Hospital-Kaohsiung Medical Center, Kaohsiung, Taiwan [CMRPG 860031, CZRPG 880253]
To study differences in the development of immunity, leukocytes from cord blood are often compared with those from adult peripheral blood. Western blot analysis is a common method for detecting proteins. In this study, we investigated the reliability of using different housekeeping proteins (beta-actin, beta-tubulin, and glyceraldehyde-3-phosphate dehydrogenase [GAPDH]) as internal controls for different leukocyte subpopulations from infants, children, and adults. Our results showed that the expression levels of beta-actin and beta-tubulin were much lower in cord blood leukocytes than in adult leukocytes, and this expression pattern persisted in children up to 3 years old. Further study revealed that the beta-actin expression level in newborns was especially lower in CD14-positive monocytes. However, cord blood and adult peripheral blood monocytes had similar expression levels of beta-actin messenger RNA (mRNA). Further experiments showed that posttranslational regulation was responsible for the low beta-actin expression level in neonatal monocytes. Thus, researchers should carefully assess the appropriate use of housekeeping gene-encoded proteins as internal standards to normalize samples for comparisons of different leukocyte populations from subjects of different ages. In this study, we determined that GAPDH was a more reliable internal control than others in Western blot analysis for comparing the development of immunity among infants, children, and adults. (C) 2011 Elsevier Inc. All rights reserved.
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