FLUOROMETRIC-DETERMINATION OF TOTAL MESSENGER-RNA WITH OLIGO(DT) IMMOBILIZED ON MICROTITER PLATES

We have developed a rapid and nonradioactive method of quantifying cyt osolic mRNA from crude cell lysates by using plastic plates to which o ligonucleotides containing poly(dT) sequences were previously immobili zed. Captured mRNA on the plate was mixed with Yoyo-1 fluorescent indi cator dye, and the resulting Yoyo-1 fluorescence of the mRNA-Yoyo-1 co mplex was measured in a fluorometer. Because Yoyo-1 signals were linea rly 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 co mparing their Yoyo-1 fluorescence with that of known concentrations of calibrator mRNA. Using this system, we found that the amount of cytos olic 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 < 0.0 1) 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 biolog y research and diagnostics.