Colorimetric ELISA measurement of specific mRNA on immobilized-oligonucleotide-coated microtiter plates by reverse transcription with biotinylated mononucleotides.
In this study, alpha-globin mRNA was captured on plastic plates to which alpha-globin-specific oligonucleotides had been covalently immobilized; the captured mRNA was immediately reverse-transcribed into cDNA in the presence of biotinylated dUTP. Addition of alkaline phosphatase-conjugated streptavidin to react with the biotin was followed by addition of substrate (p-nitrophenyl phosphate), and the resulting colorimetric development was measured at 405 nm. Because multiple biotin molecules are incorporated into cDNA during reverse transcription, and because synthesized cDNA is more stable than mRNA, we could successfully quantify by conventional colorimetry the amount of alpha-globin mRNA on the plate within a linear range of 100 pg to 10 ng. This is more sensitive than conventional Northern blotting. Therefore, the present method may be applicable to measurements of specific mRNAs in various test materials.
Tominaga
K
K
Department of Pathology, University of California, Irvine, USA.
Miura
Y
Y
Arakawa
T
T
Kobayashi
K
K
Mitsuhashi
M
M
D000818:Animals; D001667:Binding, Competitive; D001710:Biotin; D015152:Blotting, Northern; D003124:Colorimetry; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D005914:Globins / Q000235:genetics*; D018922:HL-60 Cells; D006801:Humans; D007700:Kinetics; D008099:Liver / Q000737:chemistry; D009693:Nucleic Acid Hybridization; D015345:Oligonucleotide Probes; D016133:Polymerase Chain Reaction; D012333:RNA, Messenger / Q000032:analysis*; D012194:RNA-Directed DNA Polymerase; D011817:Rabbits; D012680:Sensitivity and Specificity
D015345:Oligonucleotide Probes; D012333:RNA, Messenger; D001710:Biotin; D005914:Globins; D012194:RNA-Directed DNA Polymerase