allowed to dry at room temperature for 5 min and immediately used for laser capture microdissection using a Veritas Microdissection sys- tem (Arcturus Bioscience, Mountain View, CA, USA). Figure 1b and c provides a visual example to demonstrate the locations from which samples were taken. As CB and OD line the root surface as a single-cell layer, the underlying cementum and attached CB, as well as the under- lying dentin and attached OD, were dissected out together to minimize cellular damage caused by placing a laser beam directly onto the cells. The dissected tissues were collected onto the CapSure Macro LCM cap (Arcturus Bioscience). The collected tissue together with the mem- brane on the cap were detached from the cap and put into a 0.5 mL microcentrifuge tube containing 50 mL of PicoPure lysis buffer and the tube was incubated at 42 6C for 30 min, and processed for RNA isolation according to the manufacturer’s instructions (Arcturus Bioscience). The RNA integrity number (RIN) was measured to evaluate the quality of each RNA sample, using an Agilent 2100 Bioanalyzer and RNA 6000 Pico Kit according to the manufacturer’s instructions (Agilent Technologies, Santa Clara, CA, USA). The con- centration of total RNA was quantified by UV absorption at 260 nm using a NanoDrop 1000 spectrophotometer (Thermo Fisher Scienti- fic, Wilmington, DE, USA (Technical Support); Thermo Fisher Scientific, Waltham, MA, USA (Headquarters)). Gene expression microarray analysis Fifty nanograms of each total RNA sample were used for RNA amp- lification using Ovation Pico WTA System (NuGEN Technologies, San Carlos, CA, USA). Each amplified antisense cDNA product was converted into a sense cDNA transcript (ST-cDNA) and fragmented using WT-Ovation Exon Module (NuGEN Technologies). Each frag- mented cDNA product was biotinylated, using an Encore Biotin Module according to the manufacturer’s instructions (NuGEN Technologies). Each biotinylated probe was hybridized onto a GeneChip Mouse Gene 1.0 ST Array (Affymetrix, Santa Clara, CA, USA) for 16 h at 456C. After hybridization, the array chips were washed, stained with streptavidin phycoerythrin using an Affymetrix Fluidics Station 450 and imaged using an Affymetrix Gene Chip Scanner 3000 (Affymetrix). Real-time reverse transcriptase polymerase chain reaction (RT-PCR) RT-PCR was used to verify mRNA expression of specific tissue- related genes using the LightCycler 480 system (Roche Diagnostics, Mannheim, Germany). Twenty nanograms of each amplified cDNA sample were used. Primers were designed by the LightCycler probe design software (Roche Diagnostics, Indianapolis, IN, USA). Primer sequence analysis using a Basic Local Alignment Search Tool and melting curve analysis of the PCR products were used to ensure spe- cificity of each primer pair. Primer sequences used in this study were as follows: cytokeratin 14 (Krt14): sense 59-GCGGGATCAGTACGAG-39 antisense 59-AGTAACGGCCTTTGGT-39 bone morphogenetic protein 2 (Bmp2): sense 59-CCCGATCACCTCTCTT-39 antisense 59-ACCGCAGTCCGTCTAA-39 fibroblast growth factor 10 (Fgf10): sense 59-CCAGAGGGACCCTTAC-39 antisense 59-GGTTAAGCCCCAGGGA-39 Laser capture microdissection for root study JX Sun et al bone sialoprotein (Bsp): sense 59- GAGACGGCGATAGTTCC-39 antisense 59- AGTGCCGCTAACTCAA-39 osteocalcin (Ocn): sense 59- TGAACAGACTCCGGCG-39 antisense 59- GATACCGTAGATGCGTTTG-39 glyceraldehyde 3-phosphate dehydrogenase (Gapdh) as a house keep- ing gene: sense 59-TCCACCACCCTGTTGCTGTA-39 antisense 59-ACCACAGTCCATGCCATCAC-39 Using the LightCycler 480 software 1.5, the relative quantification of gene expression level for target vs. Gapdh (housekeeping gene) was performed (Roche). Amplification efficiency was determined for each pair of primers. Statistical analyses The robust multichip average (RMA) method was used for back- ground adjustment, quantile normalization and median polish summarization using Affymetrix Expression Console software. Significance analysis of microarrays was used for statistical analyses of microarray data (http://www-stat.stanford.edu/,tibs/SAM/). Student’s t-test was used for comparing the differential expression of each gene. P,0.05 was considered statistically significant. RESULTS RNA integrity Using the protocol described in the ‘Materials and methods’ section, we were able to obtain a sufficient quality and quantity of RNA samples for global gene expression analyses using whole genome expression micro- arrays (Affymetrix mouse gene 1.0 ST). The RIN of each sample is shown in Table 1. RINs of all RNA samples were above or close to 6. Instead of using traditional in vitro transcription from cDNA carrying the T7 promoter, we used Ribo-SPIA reactions with a combination of universal RNA primer, DNA polymerase and RNAse to isothermally amplify total RNA (Ovation Pico WTA System; NuGEN Techno- logies). Using this technique, we found that RNA samples with RIN above or close to 6 can be obtained and used successfully for whole genome microarray (Affymetrix mouse gene 1.0 ST). Quality of gene expression data from microarrays Data for quality control metrics of each array are shown in Table 2. Every array passed all quality control metrics. The area under the curve value for a receiver operating characteristic (ROC) curve plotted detection of positive controls against false detection of negative con- trols (Pos_vs_neg_auc) ranged from 0.84 to 0.89 for all arrays, indi- cating good array hybridization and signal quality for each gene expression microarray chip. Arrays were neither dim nor denoted as an outlier (Table 2 and Figure 2). After RMA expression, values were computed, and background correction and relatively identical distri- bution of expression values across arrays were ensured, indicating good array data quality. Table 1 RNA integrity numbers Samples 1 2 3 Lingual cervical loop 7.0 7.0 6.4 Apical dental follicle 6.8 8.5 3.6 Apical dental papilla 8.0 7.1 8.5 Odontoblasts Cementoblasts 6.4 6.4 5.4 6.3 6.5 5.4 9 International Journal of Oral Science