Isolation of erythroid cells from the mouse embryonic yolk sac by laser capture microdissection and subsequent microarray hybridization.
Erythropoietic tissues are complex, containing both erythroid and other cells. The embryonic yolk sac in particular contains primitive erythroid cells in low abundance. Laser capture microdissection (LCM) was performed to isolate erythroid cells, and epithelial cells, from mouse embryonic day 10 (E10) yolk sac. Quantitative RT-PCR was performed to confirm that enriched cell populations were obtained. epsilony- and betaH1-globin mRNAs were enriched in the erythroid compared to the epithelial fraction, and villin mRNA was enriched in the epithelial compared to the erythroid fraction. RNA isolated from the microdissected erythroid cells was of high quality as indicated by capillary electrophoresis. The RNA from the LCM erythroid fraction was linearly amplified with T7 RNA polymerase and hybridized to a Mouse 430A 2.0 Affymetrix array. Forty-eight percent of genes were present in the microarray assays, including low abundance transcripts such as erythroid transcription factors and enzymes involved in heme synthesis. With the LCM/microarray strategy, it will be possible to identify genes that are differentially regulated in native primitive and definitive erythroid cells.
Redmond
Latasha C
LC
Department of Human Genetics, Virginia Commonwealth University, Richmond, Richmond, VA 23298, USA.
Haar
Jack L
JL
Giebel
Mary L
ML
Dumur
Catherine I
CI
Basu
Priyadarshi
P
Ware
Joy L
JL
Lloyd
Joyce A
JA
D000818:Animals; D002469:Cell Separation / Q000379:methods*; D004622:Embryo, Mammalian / Q000166:cytology; D004847:Epithelial Cells / Q000166:cytology; D015672:Erythroid Precursor Cells / Q000166:cytology*; D020869:Gene Expression Profiling; D051379:Mice; D042282:Microdissection / Q000379:methods*; D020411:Oligonucleotide Array Sequence Analysis; D012333:RNA, Messenger / Q000032:analysis; D015017:Yolk Sac / Q000166:cytology*
D012333:RNA, Messenger