Alteration of gene expression in mice after glaucoma filtration surgery

To clarify the early alterations of gene expression using a mouse model of glaucoma filtration surgery, we carried out microarray expression analysis. Using BALB/c mice, a filtration surgery model was made by incision of the limbal conjunctiva, followed by the insertion of a 33G needle tip into the anterior chamber, and 11-0 nylon sutures. Subgroups of mice were treated intraoperatively with 0.4 mg/ml mitomycin-C (MMC). At day 3 after surgery the bleb was maintained. The bleb region tissue was sampled 3 days after the filtration surgery, and gene expression analysis was carried out using a mouse Agilent 8 × 60 K array. We found 755 hyperexpressed transcripts in the bleb region compared to control conjunctiva. The hyperexpressed transcripts included epithelial cell metaplasia-related (Il1b, Krt16, Sprr1b), inflammation-related (Ccl2, Il6) and wound healing-related (Lox, Timp1) genes. We also found downregulation of a goblet cell marker gene (Gp2) in the bleb conjunctiva. MMC treatment suppressed elastin (Eln) gene expression and enhanced keratinization-related gene expression (Krt1, Lor) in the bleb region. Our results suggest the importance of epithelial wound healing after filtration surgery, and this filtration surgery model will be a useful tool for further pathophysiological analysis.

Deregulated Adhesion Program in Palatal Keratinocytes of Orofacial Cleft Patients

Orofacial clefts (OFCs) are the most frequent craniofacial birth defects. An orofacial cleft (OFC) occurs as a result of deviations in palatogenesis. Cell proliferation, differentiation, adhesion, migration and apoptosis are crucial in palatogenesis. We hypothesized that deregulation of these processes in oral keratinocytes contributes to OFC. We performed microarray expression analysis on palatal keratinocytes from OFC and non-OFC individuals. Principal component analysis showed a clear difference in gene expression with 24% and 17% for the first and second component, respectively. In OFC cells, 228 genes were differentially expressed (p < 0.001). Gene ontology analysis showed enrichment of genes involved in β1 integrin-mediated adhesion and migration, as well as in P-cadherin expression. A scratch assay demonstrated reduced migration of OFC keratinocytes (343.6 ± 29.62 μm) vs. non-OFC keratinocytes (503.4 ± 41.81 μm, p < 0.05). Our results indicate that adhesion and migration are deregulated in OFC keratinocytes, which might contribute to OFC pathogenesis.

Importance of the Efficiency of Double-Stranded DNA Formation in cDNA Synthesis for the Imprecision of Microarray Expression Analysis

BACKGROUND The causes of imprecision in microarray expression analysis are poorly understood, limiting the use of this technology in molecular diagnostics. Two-dimensional strandness-dependent electrophoresis (2D-SDE) separates nucleic acid molecules on the basis of length and strandness, i.e., double-stranded DNA (dsDNA), single-stranded DNA (ssDNA), and RNA·DNA hybrids. METHODS We used 2D-SDE to measure the efficiency of cDNA synthesis and its importance for the imprecision of an in vitro transcription–based microarray expression analysis. RESULTS The relative amount of double-stranded cDNA formed in replicate experiments that used the same RNA sample template was highly variable, ranging between 0% and 72% of the total DNA. Microarray experiments showed an inverse relationship between the difference between sample pairs in probe variance and the relative amount of dsDNA. Approximately 15% of probes showed between-sample variation (P &lt; 0.05) when the dsDNA percentage was between 12% and 35%. In contrast, only 3% of probes showed between-sample variation when the dsDNA percentage was 69% and 72%. Replication experiments of the 35% dsDNA and 72% dsDNA samples were used to separate sample variation from probe replication variation. The estimated SD of the sample-to-sample variation and of the probe replicates was lower in 72% dsDNA samples than in 35% dsDNA samples. CONCLUSIONS Variation in the relative amount of double-stranded cDNA synthesized can be an important component of the imprecision in T7 RNA polymerase–based microarray expression analysis.