Isolation of high quality mRNA from a discrete cell cycle population identified using a nonvital dye and fluorescence activated sorting.
Flow cytometry (FCM) is useful for measuring DNA content as related to cell cycle position. We have extended this technology by developing a method that measures cellular DNA content using propidium iodide after permeabilization with lysolecithin. This technique maintains cell integrity such that high quality mRNA can be isolated from a sorted population. Unfixed MDA-468 cells, a human breast cancer cell line, were temporarily permeabilized with lysolecithin. A range of lysolecithin concentrations were studied in order to optimize DNA staining but minimize alterations in cell size and integrity. Cells permeabilized with lysolecithin in PBS, were stained with propidium iodide (50 micrograms/ml) in the presence of 1% bovine serum albumin, 1 mM Na2EDTA in PBS. The optimal concentration (4 micrograms lysolecithin/ml) combined staining approximately 90% of the cells for DNA, with minimal effects on cell size. Subsequently, the cells were assayed for DNA content as a measure of cell cycle position. MDA-468 cells identified as being in G1 were sorted and collected. From these, high quality mRNA could be isolated, as judged by its ability to be in vitro translated into protein of a wide range of molecular weights. This technique should be useful for molecular studies requiring discrete cell populations based on DNA content and/or cell cycle position.
Church
J G
JG
Terry Fox Cancer Research Laboratories, Faculty of Medicine, Memorial University of Newfoundland, St. John's, Canada.
Stapleton
E A
EA
Reilly
B D
BD
D001943:Breast Neoplasms / Q000473:pathology; D002453:Cell Cycle; D002469:Cell Separation / Q000379:methods*; D004247:DNA / Q000032:analysis*; D005434:Flow Cytometry / Q000379:methods*; D006801:Humans; D008244:Lysophosphatidylcholines; D011419:Propidium; D012333:RNA, Messenger / Q000032:analysis / Q000302:isolation & purification*; D013194:Staining and Labeling; D014407:Tumor Cells, Cultured
D008244:Lysophosphatidylcholines; D012333:RNA, Messenger; D011419:Propidium; D004247:DNA