10 Laser capture microdissection for root study JX Sun et al Robust differences in gene expression levels among tissues/cell types associated with the root formation At P,0.05, significant differences in gene expression levels were found for 1 710 genes between DP and LCL; 507 genes with differences greater than twofold (DP.LCL for 275 genes and LCL.DP for 232 genes). Expression of 1 265 genes differed significantly between DF and LCL; 327 genes with differences greater than twofold (DF.LCL for 188 genes and LCL.DF for 139 genes). Expression of 1 503 genes differed significantly between OD and CB; 416 genes with differences greater than twofold (OD.CB for 196 genes and CB.OD for 220 genes) (Figure 3). After adjusting for false discovery rate at 1% or less using signifi- cance analysis of microarrays, differential expression of 1 014 genes among LCL, DF, DP, OD and CB was found to be significant at P,0.05. Two hundred and fifteen of these genes were expressed at higher levels in the DF and DP mesenchyme than in the LCL epithelia. These genes were also expressed at higher levels in the OD and CB than Table 2 Quality metrics of gene expression microarray samples Pos_vs_neg_auca PM_Meanb Bgrd_Meanc Samples Lingual cervical loop Apical dental follicle Apical dental papilla Root odontoblasts Root cementoblasts 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 0.87 0.85 0.86 0.87 0.86 0.88 0.85 0.86 0.86 0.86 0.85 0.89 0.86 0.86 0.84 411.74 412.57 542.95 402.88 390.64 464.59 363.71 358.40 441.69 250.25 406.85 234.75 383.68 351.12 212.35 110.09 121.19 159.21 114.89 113.54 135.82 106.72 99.52 125.33 75.45 104.08 78.08 107.51 96.45 71.63 Figure 2 Box plots of log expression signals from all probes in each array after computing the RMA. Samples: LCL epithelium, apical DF and DP, OD and CB. All arrays had relatively similar distribution of expression values after RMA. No outliers were detected. CB, cementoblasts; DF, dental follicle; DP, dental papilla; LCL, lingual cervical loop; OD, odontoblasts; RMA, robust multichip average. a Ratio for detection of positive controls (probes for exons of 100 housekeeping genes) vs. false detection of negative controls (probes for introns of the same 100 housekeeping genes). This metric indicates overall microarray data quality. Typical ratios range between 0.8 and 0.9, with a value of 1.0 being perfect and a value of 0.5 showing no discernable difference between the positive and negative controls. A value significantly below 0.8 strongly indicates poor quality microarray data. b Mean signal of all perfect match probes (oligonucleotide probes, typically 25-mers containing canonical sequence specific for each gene). These are raw signal intensities before normalization. c Mean signal of background derived from antigenomic probes, which are designed not to hybridize to any genomic sequence in most studied organisms such as human, mouse, fly and etc. There are about 1000 probes for each level of GC content (0–25). International Journal of Oral Science Figure 3 Volcano plots for differential expression of genes in LCL epithelia, adjacent DP mesenchyme, DF mesenchyme, root OD and CB. Unpaired t-tests were used to compare differences in gene expression. Genes with statistically significant difference at P,0.05 and FC (fold change) o2 are shown in red, while those with Po0.05 or FC,2 are shown in grey or black (not statistically signifi- cant). CB, cementoblasts; DF, dental follicle; DP, dental papilla; LCL, lingual cervical loop; OD, odontoblasts.