Gene expression in a pure population of odontoblasts isolated by laser-capture microdissection.
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 alpha1(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.
Hoffmann
M
M
Department of Orthodontics, University of Texas at Houston Health Science Center, Dental Branch, 77030, USA.
Olson
K
K
Cavender
A
A
Pasqualini
R
R
Gaikwad
J
J
D'Souza
R N
RN
D000818:Animals; D002469:Cell Separation / Q000295:instrumentation / Q000379:methods*; D024042:Collagen Type I / Q000096:biosynthesis / Q000235:genetics; D003771:Dental Papilla / Q000166:cytology; D003810:Dentinogenesis / Q000235:genetics; D016326:Extracellular Matrix Proteins; D020869:Gene Expression Profiling / Q000379:methods*; D017403:In Situ Hybridization; D007834:Lasers; D051379:Mice; D008810:Mice, Inbred C57BL; D009804:Odontoblasts / Q000166:cytology / Q000378:metabolism; D015675:Osteocalcin / Q000096:biosynthesis / Q000235:genetics; D010750:Phosphoproteins; D011498:Protein Precursors / Q000096:biosynthesis / Q000235:genetics; D012313:RNA / Q000032:analysis; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012795:Sialoglycoproteins; D000099311:Dentin Sialophosphoprotein
D024042:Collagen Type I; D016326:Extracellular Matrix Proteins; D015675:Osteocalcin; D010750:Phosphoproteins; D011498:Protein Precursors; D012313:RNA; D012795:Sialoglycoproteins; D000099311:Dentin Sialophosphoprotein