Microarray data resulting from the Cy3 and Cy5 channels of each slide were always treated as single channel data, rather than two-channel data, facilitating more flexible analysis of experimental data. The data was analyzed as if derived from single colour arrays by combining input from individual arrays from any one slide. At least triplicate slides were used at each time-point investigated. In this study median net fluorescence intensities were initially converted to log2 scale. The log2 values were subjected to data normalization and transformation to diminish contributions from non-biological and biological variation, making signal intensities comparable across arrays, and achieving approximate normality and constant variance for subsequent statistical modelling. Typically the microarray data were subjected to z-score normalization, often followed by median subtract normalization or scaling between 0 and 1.Cluster analysis (k-means-, hierarchical clustering and self-organizing maps) are used to decrease the dimensionality of microarray data enabling isolation of genes showing similar time-course expression profiles during tooth development. Genes with a time-course of expression significantly similar to that of a pre-selected gene(s) throughout the time-course can also be found using the Profile Search feature in Spotfire v.9 Microarray Analysis Software. The expression pattern of the pre-selected gene(s) is called the master profile. The Profile Search tool calculates the similarity to this selected gene profile for all genes in the microarray data.
The microarrays were scanned and the Cy5 and cy3 fluorescence signals quantitated using the scanner standard settings. (Parameters: Scanning hardware = ScanArray Lite [PerkinElmer], Scanning software = ScanArray Express [PerkinElmer])
The hybridization protocol for the 3DNA Array 900 expression Array Detection kit for Microarrays(Genisphere Inc. 2004)was used. (Parameters: Chamber type = OTHER: hybridazation chamber, Quantity of label target used = 2, Mass unit = Micro gram, time = 18, Tiny time unit = hours, Volume = 54.8, Volume unit = Micro litre, temperature = 62)
Pregnant CD1 mice were sacrificed by cervical dislocation, the pups by decapitation. Tooth germs were isolated from mouse embryos and pups at 24hr intervals starting at 11.5 days post coitum (E11.5) ending at 7 days 2 days postnatal (P6). The day of vaginal plug was set to 0.5dpc. Embryos were staged according to the Theiler criteria5. First, right hand side, mandibular molar tooth germs were micro-dissected from ten phenotypical embryos/pups at the various developmental stages. Most of the tooth follicle remained with the tooth germ. Embryos and pup heads were immediately after removal immersed in RNAlater diluted 1:1 with PBS (Ambion Inc., TX, USA). Subsequent dissection of tooth germs was also carried out with heads immersed in diluted RNAlater. The dissected first right mandibular molar germs was immediately transferred into undiluted RNAlater™.
1ug total RNA was used in step1 of Genisphere 3DNA Array 900 Expression Array Detection Kit. 0.33ug total RNA from each of three different mice were pooled(total amount of 0.99ul~1ug) total RNA.
The RNeasy Mini protocol(Qiagen GmBh, Germany, 2004)was used for RNA isolation. Tooth germs were stored in RNAlater used subsequently for RNA isolation using the RNeasy Mini protocol. Tooth germs were homogenized using an Ultraturrax homogenizer. (Parameters: Extracted product = total_RNA, Amplification = none)
3DNA Array 900 expression Array Detection kit for Microarrays(Genisphere Inc. 2004)was used to label RNA. (Parameters: Amount of nucleic acid labeled = 1, Mass unit = Micro gram, Label used = Cy5, Amplification = none)
3DNA Array 900 expression Array Detection kit for Microarrays(Genisphere Inc. 2004)was used to label RNA. (Parameters: Amount of nucleic acid labeled = 1, Mass unit = Micro gram, Label used = Cy3, Amplification = none)