Blood. 2006 March 1; 107(5): 2170–2179.
Gene array analysisTotal RNA was isolated from passage 1 (young p1) and passage 5 (aged p5) MEFs with a RNeasy mini kit (Qiagen, Venlo, The Netherlands). During cDNA synthesis, samples were labeled with [33P]-deoxycytidine triphosphate (dCTP; MP Biochemicals, Irvine, CA). Mouse filter gene arrays (GF400a; Research Genetics, Invitrogen) were hybridized and analysis was performed as described previously.34 Expression patterns were verified by reverse transcription-polymerase chain reaction (RT-PCR).
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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],
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Affymetrix GeneChip technology and bio-informatics RNA was purified using the RNeasy Mini Kit (QIAGEN, Valencia, CA) followed by on-column DNA digestion with RNase-Free DNase (QIAGEN). RNA integrity was confirmed using the RNA 6000 Nano LabChip (Agilent Biotechnologies, Palo Alto, CA). Two replicate cRNA targets were generated from each HEC-1-A sub-line RNA preparation; all eight targets were made at the same time. Total cellular RNA (8 ug) was converted to cDNA using a T7-(deoxythymidine)24 primer and Superscript II (Life Technologies, Inc., Gaithersburg, MD). Resulting cDNA was used with the ENZO BioArray High Yield RNA Transcript labeling kit (ENZO, Farmingdale, NY) to synthesize biotin-labeled cRNA; the latter was purified on an RNeasy spin column (QIAGEN) and chemically fragmented to a size range of 35 to 200 bp. cRNAs were concurrently hybridized to HG-U133A GeneChips (Affymetrix, Santa Clara, CA). Hybridizations were performed for 16 hours, followed by incubations with streptavidin-conjugated phycoerythrin, and polyclonal anti-streptavidin antibody coupled to phycoerythrin. GeneChips were scanned using an Agilent GeneArray laser scanner and images analyzed using Affymetrix MAS 5.0 software. Bacterial sequence-derived probes on the arrays served as external controls for hybridization, whereas the housekeeping genes β-actin and GAPDH served as endogenous controls and for monitoring the quality of the RNA targets. Unsupervised nearest-neighbor hierarchical clustering (Spotfire DecisionSite, Somerville, MA) identified a significant effect of culture condition/date of RNA collection on overall gene expression profiles. Therefore, to identify candidate KLF9 gene targets, the following was separately performed on the combinations of 4S/2AS and 9S/3AS. Intensity values of probe sets were imported into GeneSpring Gx 7.3 software for analysis. Values were processed using the Robust Multiarray Analysis algorithm for background adjustment, normalization and log2-transformation of perfect match values. Data were subjected to per-chip and per-gene normalization and analyzed for differences between cell lines (fold-change, S relative to AS, value of 1.3 or higher; P < 0.05, Student's t test). Transcripts that passed these filters for both the 4S/2AS and 9S/3AS cell line combinations comprised the final KLF9-regulated gene lists and were compared for gene overlaps. The final gene list (comprised only of overlapping transcripts) was annotated using NETAFFX [24] and NCBI Entrez [25]. The microarray data have been deposited in Gene Expression Omnibus [26] as series GSE11855.
