Laser capture microdissection for root study JX Sun et al 11 Figure 4 Comparison of mRNA expression data from microarray vs. RT-PCR. Genes known to be differentially expressed were selected and compared. mRNA expression levels derived from microarrays (a) were normalized by using the RMA method and those derived from quantitative RT-PCR (b) were normalized to the expression level of house keeping gene Gapdh as described in the ‘Materials and methods’ section. For LCL epithelium, Krt14; for dental mesenchyme including DF and DP, Bmp2 and Fgf10; for CB, Bsp; and for OD, Ocn. Differential expression patterns of these genes in LCL, DF, DP, CB and OD were generally consistent between microarray and RT-PCR data, though differences in measured gene expression levels between tissue/cell types were more robust in the data from RT-PCR than those from microarray. Bmp2, bone morphogenetic protein 2; Bsp, bone sialoprotein; Fgf10, fibroblast growth factor 10; Gapdh, glyceraldehyde 3-phosphate dehydrogenase; Krt14, cytokeratin 14; Ocn, osteocalcin; OD, odontoblasts; RMA, robust multichip average; RT-PCR, reverse transcriptase polymerase chain reaction. in the DF and DP. In contrast, expression levels of 268 genes were greater in the LCL epithelial than in the mesenchymal cells including DF, DP, OD and CB. Seventeen genes were expressed at higher levels and more specifically in CB than in OD and other cells, while 26 genes were expressed higher and more specifically in OD than in CB and other cells. Comparison of gene expression data from microarrays vs. RT-PCR Expression patterns of genes known to be selective to dental epithe- lium vs. mesenchyme were examined to confirm specificity of cell isola- tion and to validate the microarray data. These genes include Krt14 for dental epithelium; Bmp2 and Fgf10 for early/undifferentiated dental mesenchyme (DF and DP); Ocn and Bsp for late/mineralized tissue forming mesenchyme (OD and CB). Expression data of these genes derived from microarrays and from RT-PCR were compared (Figure 4). Krt14, an established epithelial cell marker, was expressed mainly in the LCL epithelium. In contrast, expression of Bmp2 and Fgf10 was greater in the adjacent DP than in the LCL and DF. Both OD and CB expressed Bsp and Ocn; however, Bsp was expressed at higher levels in CB than in OD, while Ocn was expressed at higher levels in OD than in CB, consistent with the previous analysis of these tissues by in situ hybridization.33 DISCUSSION The techniques for LCM presented in this study were optimized to allow histological identification of odontogenic epithelia, mesenchy- me and their lineage cells in the mineralized structures of teeth and mandibles while preserving the integrity of cellular component, par- ticularly RNA. We demonstrated techniques for sectioning of unfixed and undecalcified frozen mouse mandibles, alcohol-based staining, LCM, RNA isolation, RNA amplification, microarray processing and subsequent gene expression data analyses. High-quality microar- ray data were noted, with high gene detection rates and signal-to-noise ratios (mean signals from perfect match probes vs. antigenomic probes/background) for all microarray chips processed. Further, high agreement rates were observed between gene expression data from microarrays and RT-PCR. These gene expression data also agreed with the limited findings from other studies using different techniques, showing success of our protocol presented here.13,34–38 Preservation of unfixed and undecalcified hard tissue samples du- ring sectioning is challenging. Previous studies have shown a tech- nique mounting these samples on transparent adhesive films to maintain tissue integrity.39–40 However, special equipment, materials and skills are required for this technique. Further, xylene cannot be used to completely dry the tooth tissues on the tapes. Using only a series of increasing concentration of alcohol to dry the tissues is fre- quently inadequate. The remaining water in the tissues increases the risk of RNA degradation from any residual/contaminated RNAses and also compromises the cutting ability of the laser beam during micro- dissection. By removing the unnecessary part of hard tissue, including the erupted part of mandibular incisors, surrounding mandibular bones and condyles, we showed that a conventional frozen section technique can be used to obtain sections of developing roots with well-preserved tissue integrity and histological structures. Mammalian tissues are heterogenous, with several cell phenotypes and with various stages of differentiation within a given sample, pre- senting a challenge for in vivo molecular analyses. The LCM technique offers a superior way to collect target cells and minimize contamina- tion with unwanted cells. During the LCM procedure, RNA degrada- tion increases as processing time and procedures increase.41 RNAlater, an RNA stabilization reagent, was shown to improve RNA quality from the LCM samples.42–43 Using alcohol-based cresyl violet staining International Journal of Oral Science