Fluorometric determination of total mRNA with oligo(dT) immobilized on microtiter plates.
We have developed a rapid and nonradioactive method of quantifying cytosolic mRNA from crude cell lysates by using plastic plates to which oligonucleotides containing poly(dT) sequences were previously immobilized. Captured mRNA on the plate was mixed with Yoyo-1 fluorescent indicator dye, and the resulting Yoyo-1 fluorescence of the mRNA-Yoyo-1 complex was measured in a fluorometer. Because Yoyo-1 signals were linearly increased in proportion to the amount of applied mRNA in the range 10-250 ng, the amount of mRNA in test samples can be determined by comparing their Yoyo-1 fluorescence with that of known concentrations of calibrator mRNA. Using this system, we found that the amount of cytosolic mRNA in undifferentiated U937 and HL-60 cells was 268.6 +/- 13.1 and 282.0 +/- 7.8 ng/10(6) cells, respectively, significantly (P < 0.01) more than that of phorbol ester-induced differentiated U937 and HL-60 cells (145.3 +/- 13.9 and 164.7 +/- 11.6), respectively. Therefore, the present system may be applicable to both medical molecular biology research and diagnostics.
Department of Pathology, University of California Irvine.
Y
Y
Miura
Y
Y
Ichikawa
T
T
Ishikawa
M
M
Ogura
R
R
de Fries
H
H
Shimada
M
M
Mitsuhashi
D000818:Animals; D001583:Benzoxazoles; D015152:Blotting, Northern; D002454:Cell Differentiation / Q000187:drug effects; D002460:Cell Line; D016895:Culture Media, Serum-Free; D003600:Cytosol / Q000737:chemistry; D005456:Fluorescent Dyes; D005914:Globins / Q000235:genetics; D018922:HL-60 Cells; D006801:Humans; D007700:Kinetics; D007963:Leukocytes, Mononuclear / Q000737:chemistry / Q000648:ultrastructure; D009693:Nucleic Acid Hybridization; D009838:Oligodeoxyribonucleotides; D011806:Quinolinium Compounds; D012333:RNA, Messenger / Q000032:analysis*; D011817:Rabbits; D013050:Spectrometry, Fluorescence; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology
D001583:Benzoxazoles; D016895:Culture Media, Serum-Free; D005456:Fluorescent Dyes; D009838:Oligodeoxyribonucleotides; D011806:Quinolinium Compounds; D012333:RNA, Messenger; C027903:oligo (dT); C075296:1,1'-((4,4,7,7-tetramethyl)-4,7-diazaundecamethylene)bis-4-(3-methyl-2,3-dihydro(benzo-1,3-oxazole)-2-methylidene)quinolinium; D005914:Globins; D013755:Tetradecanoylphorbol Acetate