Flow cytometric estimation of poly(A)+ RNA by fluorescent in situ hybridization.
A method for the detection of poly(A)+ RNA in cell suspensions by in situ hybridization and flow cytometry is described. The hybridizing properties of oligothymidylate o(dT) was well preserved after coupling to fluorescein. This fluorescent oligonucleotide was used as a probe to determine the poly(A)+ RNA content of fixed HL60 leukemic cells by flow cytometry. Labeling was considerably reduced by treating the cells with RNAse, and by competitive hybridization with either poly(U) or free poly(A). Labeling was also decreased in a time-dependent fashion by incubating the cells with actinomycin D prior to fixation. This method represents an improvement on the methods measuring total RNA and could be of value in investigations on the effect of drugs on RNA metabolism.
Belloc
F
F
Laboratoire d'Hématologie, Hôpital Haut Lévêque, Pessac, France.
Lacombe
F
F
Dumain
P
P
Mergny
J L
JL
Lopez
F
F
Bernard
P
P
Reiffers
J
J
Boisseau
M R
MR
D003609:Dactinomycin / Q000494:pharmacology; D005434:Flow Cytometry / Q000379:methods*; D006801:Humans; D017403:In Situ Hybridization / Q000379:methods*; D015345:Oligonucleotide Probes; D011061:Poly A / Q000032:analysis*; D011071:Poly T / Q000737:chemistry; D012333:RNA, Messenger / Q000032:analysis*; D012680:Sensitivity and Specificity; D014407:Tumor Cells, Cultured / Q000737:chemistry / Q000187:drug effects
D015345:Oligonucleotide Probes; D012333:RNA, Messenger; D003609:Dactinomycin; D011061:Poly A; D011071:Poly T