Quantitative detection of messenger RNA by solution hybridization and enzyme immunoassay.
A novel nucleic acid detection technique is described for the quantitative measurement of eukaryotic mRNA in biological samples. The procedure involves two steps: a hybridization reaction in solution with a biotinylated cDNA probe, and a conventional enzyme immunoassay that uses a monoclonal antibody for DNA.RNA hybrids to detect the specific mRNA.cDNA complexes. The method has comparable sensitivity to 32P-based methods and yields results that are quantitative and highly reproducible. Furthermore, the test can be performed using unfractionated cytoplasm without the need for extraction with organic solvents. This technique provides a rapid and quantitative method for studying changes in cellular mRNA levels, and it is suitable for testing large numbers of samples.
Department of Pediatrics, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205.
F
F
Coutlee
E A
EA
Rubalcaba
R P
RP
Viscidi
J E
JE
Gern
P A
PA
Murphy
H M
HM
Lederman
D000911:Antibodies, Monoclonal; D002460:Cell Line; D015342:DNA Probes; D004731:Endotoxins / Q000494:pharmacology; D005796:Genes; D006801:Humans; D007124:Immunoenzyme Techniques; D007202:Indicators and Reagents; D007375:Interleukin-1 / Q000235:genetics / Q000494:pharmacology; D009693:Nucleic Acid Hybridization; D012333:RNA, Messenger / Q000032:analysis* / Q000235:genetics / Q000302:isolation & purification; D011994:Recombinant Proteins / Q000494:pharmacology; D012996:Solutions; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D014407:Tumor Cells, Cultured / Q000187:drug effects / Q000378:metabolism
D000911:Antibodies, Monoclonal; D015342:DNA Probes; D004731:Endotoxins; D007202:Indicators and Reagents; D007375:Interleukin-1; D012333:RNA, Messenger; D011994:Recombinant Proteins; D012996:Solutions; D013755:Tetradecanoylphorbol Acetate