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Modified house‐keeping gene expression in a rat tail compression loading‐induced disc degeneration model
Author(s) -
Yurube Takashi,
Takada Toru,
Hirata Hiroaki,
Kakutani Kenichiro,
Maeno Koichiro,
Zhang Zhongying,
Yamamoto Junya,
Doita Minoru,
Kurosaka Masahiro,
Nishida Kotaro
Publication year - 2011
Publication title -
journal of orthopaedic research
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.041
H-Index - 155
eISSN - 1554-527X
pISSN - 0736-0266
DOI - 10.1002/jor.21406
Subject(s) - glyceraldehyde 3 phosphate dehydrogenase , messenger rna , beta 2 microglobulin , actin , lactate dehydrogenase , gene expression , lactate dehydrogenase a , housekeeping gene , biology , microbiology and biotechnology , gene , chemistry , biochemistry , immunology , enzyme
House‐keeping genes (HKGs) are generally used as endogenous controls for molecular normalization in quantitative PCR analysis. However, whether all the so‐called HKGs are useful for intervertebral disc research is controversial. Our objective was, using a prevalidated rat tail static compression loading‐induced disc degeneration model, to clarify the feasibility of common HKGs for gene‐quantification in the nucleus pulposus cells. In real‐time RT‐PCR for five HKGs [β‐actin, β‐glucuronidase, β‐2 microglobulin, glyceraldehyde 3‐phosphate dehydrogenase (GAPDH), and lactate dehydrogenase A (LDHA)], static compression at 1.3 MPa for up to 56 days demonstrated messenger RNA (mRNA) expression levels of consistent β‐2 microglobulin and GAPDH, slightly up‐regulated β‐glucuronidase, and fairly down‐regulated β‐actin and LDHA. Especially, β‐actin had a drastic suppression to 0.15‐fold in the loaded relative to unloaded discs at 7 days. In immunofluorescence, β‐actin showed a significant down‐regulation to almost undetectable levels from 28 days, while GAPDH was constantly detected throughout. β‐Actin mRNA and protein‐distribution are thought to be affected by the loading treatment, however, GAPDH mRNA and protein‐distribution can retain relatively stable expressions. Under prolonged static compression, β‐actin and probably LDHA are inappropriate, and GAPDH is a feasible HKG as internal references in the disc cells. © 2011 Orthopaedic Research Society Published by Wiley Periodicals, Inc. J Orthop Res 29: 1284–1290, 2011