Analysis of Osteopontin in Mouse Growth Plate Cartilage by Application of Laser Capture Microdissection and RT-PCR

Abstract

Gene expression of osteopontin (OPN) has been investigated in mice by application of laser capture microdissection (LCM) and reverse transcriptase-polymerase chain reaction (RT-PCR) analysis. LCM permits individual cells to be isolated ("captured") from tissue sections for molecular analyses. In this study, chondrocytes were captured from growth plate zones in frozen sections of tibiae from 1-11-day-old postnatal mice. RNA was extracted from cells, DNAse-treated, and reverse-transcribed. cDNA was amplified by PCR and OPN mRNA was revealed on agarose gels. Whole cartilage and brain (a positive control) from the same animals also were examined. Reactions containing no RT were negative controls, and 18S rRNA standardized expressed message from captured cells. RT-PCR analysis of laser-captured whole cartilage showed a general qualitative loss of OPN mRNA as animal age increased. Youngest mice gave equivalent OPN expression over all laser-microdissected cartilage zones. For 7-11 day-old mice, OPN expression was qualitatively greatest in resting and lowest in hypertrophic regions of cartilage. Expression of OPN correlated with mineral in the tissue suggests that OPN functionally may inhibit normal vertebrate growth plate mineralization, and its loss with increasing tissue maturation appears permissive to mineral development.