cDNA microarray

We used a custom-made utero-placental cDNA microarray that was developed in our laboratory as previously described [21,22]. In brief, a cDNA library was constructed from mRNA isolated from endometrial (caruncular and intercaruncular endometrium) and placental tissues (cotyledonary and intercotyledonary fetal membrane) of Japanese black cows. The PCR products of about 4,000 clones from the cDNA library were robotically spotted onto glass slides. The clones were simultaneously sequenced using the MegaBACE 1000 DNA Sequencing System (Amersham Pharmacia Biotech, Piscataway, NJ). The array contained 3,955 spots that were clustered into 1,738 unique genes on the basis of sequence analysis. An additional 35 genes that were not included in the cDNA library but had also been spotted onto the cDNA microarray were used for the analysis since these genes have been shown to be characteristically expressed during gestation in bovines and humans [4,11,19,23-27]. MMP related genes made up the bulk of this group. The cDNA microarray hybridization procedures were described in previous reports [21,22]. Briefly, two μg of poly (A)+ RNA were reverse transcribed in the presence of cyanine 3 (Cy3) or Cy5 fluorescence dye (Amersham Biosciences, Buckinghamshire, UK) using SuperScript II reverse transcriptase (Invitrogen) to make the hybridization probes. Identical samples were labeled separately with either Cy3- or Cy5-dye. Thus, two hybridization reactions could be carried out with the same sample. The arrays were sequentially washed with different concentrations of SSC solutions after 16 hr incubation at 65°C. The arrays were dried by centrifugation at 1,000 × g. Hybridization images were immediately scanned by a GenePix 4000B laser scanner (Axon Instrument, Union City, CA, USA) and analyzed with GenePix Pro 4.0 software. Data normalization was performed by previously described procedures [26,27]. The local background intensity of each array spot was smoothed by local weight regression (Lowess) and subtracted from the spot intensity data. The subtracted intensity data were subjected to non-parametric regression and local variance normalization since non-parametric regression can reduce intensity-dependent biases. The accuracy is improved over that of linear regression if the points in the scatter plot of Cy3 versus (vs.) Cy5 are not distributed around a straight line. Data for individual genes were obtained by averaging the intensity values of analogous spots on the microarray. Data were log₂ transformed and used for cluster analysis. The Cluster 3.0 program was used for the hierarchical clustering. The hierarchically clustered data were visualized using the TreeView 0.99 program (M.B. Eisen's based clustering program [28],