High-quality RNA and DNA from flow cytometrically sorted human epithelial cells and tissues.
Microarray technologies have made possible comprehensive analyses of nucleic acid sequence and expression. However, the technology to obtain efficiently high-quality RNA and DNA suitable for array analysis from purified populations of neoplastic cells from human tissues has not been well addressed. Microdissection can enrich for populations of cells present in various tumor tissues, but it is not easily automated or performed rapidly, and there are tissues in which cells of interest cannot be readily isolated based on morphologic criteria alone. Here we describe a protocol for efficient RNA and DNA isolation from flow cytometrically purified whole epithelial cells from primary tissue. The aqueous reagent, RNAlater, which preserves RNA, allows immunolabeling and purification of whole epithelial cells by flow sorting without special instrument preparation to reduce RNase activity. We used real-time PCR to determine RNA quality afterflow sorting. High-quality RNA and DNA suitable for expression and genotype analysis can be readily obtained from flow cytometrically purified populations of neoplastic cells from human tissues.
Barrett
M T
MT
Fred Hutchinson Cancer Research Center, Seattle, Washington, USA.
Glogovac
J
J
Prevo
L J
LJ
Reid
B J
BJ
Porter
P
P
Rabinovitch
P S
PS
D001483:Base Sequence; D001943:Breast Neoplasms / Q000737:chemistry; D002460:Cell Line; D004247:DNA / Q000032:analysis*; D004847:Epithelial Cells / Q000737:chemistry; D005260:Female; D005434:Flow Cytometry / Q000379:methods*; D005838:Genotype; D006801:Humans; D007633:Keratins / Q000032:analysis; D016133:Polymerase Chain Reaction; D012313:RNA / Q000032:analysis*
D012313:RNA; D007633:Keratins; D004247:DNA