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.


METHODS
Evaluation of differential mRNA and protein expression of Dlk, Frizzled-2, and CD-24 in fibroids compared with adjacent myometrium. University hospital.


METHODS
Five women undergoing medically indicated hysterectomy for symptomatic fibroids.


METHODS
Microarray analysis of up to 33000 genes, reverse transcriptase-polymerase chain reaction (RT-PCR), real-time RT-PCR, Western blot, and immunohistochemistry.


METHODS
Expression of mRNA transcripts and protein in fibroid compared with myometrium.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.


CONCLUSIONS
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.
Catherino
William H
WH
Pediatric and Reproductive Endocrinology Branch, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland 20892, USA.
Prupas
Cara
C
Tsibris
John C M
JC
Leppert
Phyllis C
PC
Payson
Mark
M
Nieman
Lynnette K
LK
Segars
James H
JH
D015703:Antigens, CD / Q000235:genetics* / Q000378:metabolism; D015153:Blotting, Western; D051927:CD24 Antigen; D003199:Computer Systems; D005260:Female; D051157:Frizzled Receptors; D015870:Gene Expression; D006023:Glycoproteins / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D007150:Immunohistochemistry; D007889:Leiomyoma / Q000235:genetics* / Q000378:metabolism; D008562:Membrane Glycoproteins; D009215:Myometrium / Q000378:metabolism; D020411:Oligonucleotide Array Sequence Analysis; D016133:Polymerase Chain Reaction; D012333:RNA, Messenger / Q000378:metabolism; D043562:Receptors, G-Protein-Coupled; D017981:Receptors, Neurotransmitter / Q000235:genetics* / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015398:Signal Transduction / Q000235:genetics; D014594:Uterine Neoplasms / Q000235:genetics* / Q000378:metabolism
D015703:Antigens, CD; D051927:CD24 Antigen; C497889:CD24 protein, human; C489742:FZD2 protein, human; D051157:Frizzled Receptors; D006023:Glycoproteins; D008562:Membrane Glycoproteins; D012333:RNA, Messenger; D043562:Receptors, G-Protein-Coupled; D017981:Receptors, Neurotransmitter