Analysis of odontoblast gene expression using a novel approach, laser capture microdissection.
Studying the mechanisms of molecular interactions in developing tissues demands sensitive molecular biological in vivo and in vitro techniques. Laser capture microdissection (LCM) allows for the isolation of mRNA in histological sections even from single cells, thus enabling the identification of in vivo gene expression products in closely circumscribed tissue areas. The aims of this study were to assess the optimal fixation, processing, and staining conditions to retrieve RNA from microdissected odontoblasts. Fluorometric assays and RT-PCR analysis of alpha 1(I) collagen, dentin sialophosphoprotein (Dspp), and osteocalcin (OC) confirmed that the total RNA isolated from day 0 and day 3 captured odontoblasts was sufficient in quantity and quality. Our results indicate that individual odontoblasts obtained by LCM are morphologically intact and chemically unaltered, allowing accurate molecular and biochemical analyses.
Hoffmann
Matthias
M
Department of Orthodontics, Dental Branch, University of Texas Health Science Center, 6516 John Freeman Ave., Houston, Texas 77030, USA.
Gaikwad
Joel
J
Schmalz
Gottfried
G
Cavender
Adriana
A
D'Souza
Rena
R
D000818:Animals; D004210:Dissection / Q000379:methods; D005470:Fluorometry; D015870:Gene Expression; D007834:Lasers; D051379:Mice; D009804:Odontoblasts / Q000502:physiology*; D016133:Polymerase Chain Reaction; D012313:RNA / Q000302:isolation & purification; D020133:Reverse Transcriptase Polymerase Chain Reaction
D012313:RNA