12 Laser capture microdissection for root study JX Sun et al on frozen sections instead of aqueous stains such as hematoxylin also helps preserve RNA integrity, thereby improving the subsequent gene expression analyses using RT-PCR and microarrays. The removal of water rinsing steps and the use of higher concentrations of ethanol in our modified protocol minimize endogenous or exogenous RNase activity, thereby reducing RNA degradation. Similar findings with the alcohol-based staining method were also reported for laser capture microdissection of endometrial cancer, colon and brain tissues.41,44–45 In the present study, we found that the use of alcohol-based cresyl violet staining without RNAlater application was adequate. However, the RNAlater application may be useful for routine collection of clini- cal samples and requires further investigation. Importantly, our findings of known genes at both gene expression level and tissue distribution are parallel to those described in the previous studies. Krt14 is highly expressed by odontogenic epithelial cells including cervical loop epithelia.34 Consistently, we found that Krt14 was expressed significantly higher in the LCLs of mandibular incisors than the mesenchymal counterparts. By contrast, Bmp2 and Fgf10 were expressed at significantly higher level in the pulpal mesenchyme than in the cervical loop epithelia, similar to the previous reports.13,35–36 Data from both in vitro and in vivo studies suggest that BMP2 induces the differentiation of odontogenic mesenchymal cells toward an odontoblastic lineage46–49 and may facilitate root dentin formation. FGF10 was shown to stimulate the proliferation of cervical loop epithelial cells, ectopic ameloblast differentiation and enamel formation on the lingual surface of mandibular incisors in sprouty-2 and -4 knockout mice (Spry42/2; Spry21/2 mice).35 Similar to our findings, other researchers have reported that BSP is expressed at much higher level at both protein and gene levels in CB than in OD and thus is a good marker for cementum formation.36–37 OCN is found in bone and dentin, as well as in the cells forming these two tissues (i.e., osteoblasts and OD). OCN is considered as a marker for bone maturation.38 Our microarray data are consistent with those from RT-PCR and previous findings suggesting that our LCM tech- nique presented here can be used successfully and reliably for a global study of gene expression and molecular interactions between odonto- genic epithelial and mesenchymal stem cells during tooth root forma- tion. Moreover, the technique described here should prove valuable for defining the behaviors of specific cells types associated with other mineralized tissues. Microarray analyses provide large scale gene expression data that usually require further processing. Through analyses of our microar- ray data, some novel genes were revealed that exhibited differential expression patterns during the root formation. An example was the fibulin gene family. Fibulins are a family of secreted glycoproteins related to basement membranes, elastic fibers and other matrices.50 We found that six of the seven known fibulin family members, i.e., fibulin-1, -2, -3 (also known as Efemp1, EGF-containing fibulin-like extracellular matrix protein-1), -5 (also known as DANCE, EVEC), -6 (also known as Hemicentin-1 or Hmcn1) and -7 (also known as TM14) were expressed differentially in our developing incisor and molar root samples (data not shown). Excitingly, our microarray results suggest that some of these fibulins are differentially expressed in OD and CB. Importantly, in previous studies researchers noted fibulin-1 and -2 were expressed prominently in areas undergoing epithelial–mesenchymal transitions, while the expression of fibulin- 2 is more restricted.51–52 Fibulin-3 may play a role in the formation of cartilage and bone.53 Mutations in fibulin-5 were associated with age- related macular degeneration,54 while the relationship between fibu- lin-6 and age-related macular degeneration is still controversial.55–56 International Journal of Oral Science Fibulin-5, -6 and -7 were shown to play a role in the cell attachment.57–59 Our ongoing studies are now focused on mapping the expression of this family of genes during tooth root development by in situ hybridi- zation and immunohistochemistry using sections obtained from mice at different stages of tooth root development. CONCLUSIONS We describe a method for tissue preparation, cell collection and gene expression analyses of developing teeth using laser capture microdis- section and microarrays. Using the presented protocol, we successfully performed the gene expression analyses and revealed robust gene expression changes, as well as novel genes associated with tooth root formation in mouse molars and incisors. ACKNOWLEDGEMENTS The authors would like to thank Dr Robert L Vessella and Dr Colm Morrissey in the Department of Urology, University of Washington for their technical support of LCM; Dr Theo Bammler, Dr Michael Coon and Dr Dick Beyer in the Department of Environmental and Occupational Health Sciences, University of Washington for their technical assistance in microarray analyses; Dr Brian Foster for his critical comments. This work is supported by NIH grant no. 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