Gene Expression in a Pure Population of Odontoblasts Isolated by Laser-capture Microdissection

Abstract

Studies of odontoblast differentiation and function have been limited due to difficulties in obtaining sufficient numbers of intact cells. We describe a novel approach of laser-capture microdissection to obtain homogenous populations of pre-odontoblasts and odontoblasts from tissue sections of mouse molar cusp tips. Fixation, processing, and staining conditions were assessed for the optimal retrieval of total RNA from microdissected odontoblasts. Fluorometric assays and RT-PCR analysis of α1(I) collagen, dentin sialophosphoprotein (Dspp), and osteocalcin (OC) confirmed that the total RNA from three-day-old captured odontoblasts was sufficient in quantity and quality. Odontoblast-specific gene expression was studied by RT-PCR analysis performed in a single streptavidin-coated tube. At E15.5, Days 0 and 3, gene expression in laser-captured odontoblasts resembled that seen in vivo by in situ hybridization. The use of LCM is thus a valuable means of retrieving quality RNA from discrete populations of odontoblasts at different stages of dentinogenesis.

Keywords

laser microdissection odontoblasts dentin matrix gene expression.

Get full access to this article

View all access options for this article.

References

Butler WT , Ritchie H. (1995). The nature and functional significance of dentin extracellular matrix proteins. Int J Dev Biol 39:169-179.

Cote GJ , Khorana S. , Huang E. , Gagel RF (1997). A technique for the generation of a stable and reusable source of cDNA from human blood or tissue. Biochemica 4:14-18.

D'Souza RN , Cavender A. , Sunavala G. , Alvarez J. , Ohshima T. , Kulkarni AB , et al. (1997). Gene expression patterns of murine dentin matrix protein 1 (Dmpl) and dentin sialophosphoprotein (DSPP) suggest distinct developmental functions in vivo. J Bone Miner Res 12:2040-2049.

Desbois C. , Hogue DA , Karsenty G. (1994). The mouse osteocalcin gene cluster contains three genes with two separate spatial and temporal patterns of expression. J Biol Chem 269:1183-1190.

Emmert-Buck MR , Bonner RF , Smith PD , Chuaqui RF , Zhuang Z. , Goldstein SR , et al. (1996). Laser capture microdissection. Science 274:998-1001.

Frohlich J. , Konig H. (2000). New techniques for isolation of single prokaryotic cells. FEMS Microbiol Rev 24:567-572.

Going JJ , Gusterson BA (1999). Molecular pathology and future developments. Eur J Cancer 35:1895-1904.

Hanks CT , Sun ZL , Fang DN , Edwards CA , Wataha JC , Ritchie HH , et al. (1998). Cloned 3T6 cell line from CD-1 mouse fetal molar dental papillae. Connect Tissue Res 37:233-249.

Lahr G. ( 2000). RT-PCR from archival single cells is a suitable method to analyze specific gene expression. Lab Invest 80:1477-1479.

10.

Leethanakul C. , Patel V. , Gillespie J. , Shillitoe E. , Kellman RM , Ensley JF , et al. (2000). Gene expression profiles in squamous cell carcinomas of the oral cavity: use of laser capture microdissection for the construction and analysis of stage-specific cDNA libraries. Oral Oncol 36:474-483.

11.

Linde A. , Goldberg M. (1993). Dentinogenesis. Crit Rev Oral Biol Med 4:679-728.

12.

Metsaranta M. , Toman D. , de Crombrugghe B. , Vuorio E. (1991). Specific hybridization probes for mouse type I, II, III and IX collagen mRNAs. Biochim Biophys Acta 1089:241-243.

13.

Schutze K. , Lahr G. ( 1998). Identification of expressed genes by laser-mediated manipulation of single cells. Nat Biotechnol 16:737-742.

14.

Simone NL , Bonner RF , Gillespie JW , Emmert-Buck MR , Liotta LA (1998). Laser-capture microdissection: opening the microscopic frontier to molecular analysis. Trends Genet 14:272-276.