Glyceraldehyde-3-phosphate dehydrogenase is a reliable internal control in Western blot analysis of leukocyte subpopulations from children |
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Authors: | Hong-Ren Yu Ho-Chang Kuo Hsin-Chun Huang Li-Tung Huang You-Lin Tain Chih-Cheng Chen Chi-Di Liang Jiunn-Ming Sheen I-Chun Lin Chi-Chiang Wu Chia-Yu Ou Kuender D Yang |
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Institution: | aDepartment of Pediatrics, Chang Gung Memorial Hospital–Kaohsiung Medical Center, Graduate Institute of Clinical Medical Science, Chang Gung University College of Medicine, Kaohsiung 833, Taiwan;bDepartment of Obstetrics, Chang Gung Memorial Hospital–Kaohsiung Medical Center, Graduate Institute of Clinical Medical Science, Chang Gung University College of Medicine, Kaohsiung 833, Taiwan |
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Abstract: | 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 (β-actin, β-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 β-actin and β-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 β-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 β-actin messenger RNA (mRNA). Further experiments showed that posttranslational regulation was responsible for the low β-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. |
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Keywords: | β-Actin GAPDH Internal control Monocyte Neonate Western blot |
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