Detection of oncogene expression by fluorescent in situ hybridization in combination with immunofluorescent staining of cell surface markers.
To study oncogene expression in heterogeneous cell populations we developed and optimized a non-radioactive in situ hybridization technique using biotinylated single-stranded RNA probes and combined this technique with immunofluorescent staining of cell surface markers. As a model for our studies we used HL60 cells. In these cells we detected c-myc mRNA molecules by in situ hybridization following staining of the pan myeloid cell surface marker CD33, by a monoclonal antibody. Hybrids were detected by streptavidin-FITC and CD33 by a TRITC-conjugated antibody. Controls involved pretreatment with RNAase, hybridization with sense RNA probes and blocking with an excess of unlabeled antisense probes. The integrity of the RNA in the cell was shown by hybridization with the GAPDH antisense probe. Essential for successful double-labeling was the choice of a fixation procedure that was suitable for the in situ hybridization and mild enough not to destroy the cell surface marker staining. This fluorescent in situ hybridization in combination with cell surface marker staining will be useful for studying gene expression in phenotypically well-defined cell populations.
Bakkus
M H
MH
Department of Immunology, Erasmus University, Rotterdam, The Netherlands.
Brakel-van Peer
K M
KM
Adriaansen
H J
HJ
Wierenga-Wolf
A F
AF
van den Akker
T W
TW
Dicke-Evinger
M J
MJ
Benner
R
R
D015703:Antigens, CD; D015214:Antigens, Differentiation, Myelomonocytic; D000954:Antigens, Surface / Q000032:analysis*; D005455:Fluorescent Antibody Technique; D015870:Gene Expression; D006801:Humans; D009693:Nucleic Acid Hybridization; D009857:Oncogenes; D015347:RNA Probes; D012333:RNA, Messenger / Q000032:analysis*; D063268:Sialic Acid Binding Ig-like Lectin 3
D015703:Antigens, CD; D015214:Antigens, Differentiation, Myelomonocytic; D000954:Antigens, Surface; C571163:CD33 protein, human; D015347:RNA Probes; D012333:RNA, Messenger; D063268:Sialic Acid Binding Ig-like Lectin 3