Strategy for elucidating differentially expressed genes in leiomyomata identified by microarray technology

Objective: cDNA microarray technology identifies genes that are differentially expressed between tissues. Our previous study identified several genes that might contribute to the fibroid phenotype. We therefore sought to confirm genes involved in three distinct signal transduction pathways. Design: Evaluation of differential mRNA and protein expression of Dlk, Frizzled-2, and CD-24 in fibroids compared with adjacent myometrium. Setting: University hospital. Patient(s): Five women undergoing medically indicated hysterectomy for symptomatic fibroids. Intervention(s): Microarray analysis of up to 33,000 genes, reverse transcriptase-polymerase chain reaction (RT-PCR), real-time RT-PCR, Western blot, and immunohistochemistry. Main Outcome Measure(s): Expression of mRNA transcripts and protein in fibroid compared with myometrium. Result(s): A more extensive microarray confirmed differential expression of Frizzled-2 and CD-24 but did not confirm Dlk overexpression. RT-PCR and real-time PCR demonstrated equivalent Dlk mRNA expression between fibroid and myometrium (ratio, 1.02), a slight Frizzled-2 overexpression (ratio, 2.09), and robust CD-24 overexpression in fibroids (ratio, 12.35). Western blot and immunohistochemistry confirmed Frizzled-2 overexpression, but did not confirm Dlk overexpression. Conclusion(s): Microarray technology is the first phase of tissue evaluation, but changes in gene expression must be confirmed. Confirmed genes can then be used to generate hypotheses testing their involvement in fibroid development.