TY - JOUR
T1 - Isolation of high quality mRNA from a discrete cell cycle population identified using a nonvital dye and fluorescence activated sorting
AU - Church, Jon G.
AU - Stapleton, Ernest A.
AU - Reilly, Brian D.
PY - 1993
Y1 - 1993
N2 - 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 pro‐pidium 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 μg/ml) in the presence of 1% bovine serum albumin, 1 mM Na2EDTA in PBS. The optimal concentration (4 μg lysolecithin/ml) combined staining ∼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. © 1993 Wiley‐Liss, Inc.
AB - 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 pro‐pidium 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 μg/ml) in the presence of 1% bovine serum albumin, 1 mM Na2EDTA in PBS. The optimal concentration (4 μg lysolecithin/ml) combined staining ∼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. © 1993 Wiley‐Liss, Inc.
KW - DNA staining
KW - Flow cytometry
KW - MDA‐468
KW - cell cycle
KW - cell integrity
KW - in vitro translation
KW - lysolecithin permeabilization
UR - http://www.scopus.com/inward/record.url?scp=0027523377&partnerID=8YFLogxK
U2 - 10.1002/cyto.990140306
DO - 10.1002/cyto.990140306
M3 - Article
C2 - 7682493
AN - SCOPUS:0027523377
SN - 0196-4763
VL - 14
SP - 271
EP - 275
JO - Cytometry
JF - Cytometry
IS - 3
ER -