Modified house-keeping gene expression in a rat tail compression loading-induced disc degeneration model.

Abstract

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.