Time-Resolved Fluorometric Detection of Cytokine mRNAs Amplified by RT-PCR

PCR (polymerase chain reaction)products are conventionally detected by agarose gel electrophoresis and ethidium bromide staining.However, the specificity and sensitivity of detection can be increased by hybridization with a labeled probe that is complementary to an internal sequence in the amplified target.Time-resolved fluorometry (TRF)utilizes nonradioactive lanthanide chelates with large Stoke’s shifts as the labels (1 ).For the time-resolved measurement of lanthanide fluorescence, a fluorometer is used (e.g., those manufactured by Wallac)that has a xenon flash lamp at 340-nm wavelength with 1-s intervals between flashes.After the excitation light flash and a delay time of 0.4 s, the emission from the lanthanides is measured at longer wave-lengths (613,643, and 545 nm for Eu, Sm³⁺ , and Tb³⁺ , respectively)for another 0.4 s.The excitation-emission measurement cycles are repeated 1000 � during a total measurement time of 1 s per sample. The lanthanides, e.g., europium (Eu³⁺ ), samarium (Sm³⁺ ) and terbium (Tb³⁺ ), have manifold longer fluorescence lifetimes than most biological compounds.A time delay after the excitation pulse eliminates nonspecific background by allowing the short-lived autofluores-cence of the biological material to decline to an insignificant level before measuring the specific signals from the lanthanides (2 ).This delay time is the time-resolving principle and combined together with large Stoke’s shifts, it is the critical element that makes TRF such a sensitive technique.Substances with the same fluorescence characteristics as lanthanides are rarely, if ever, found in natural samples, and background from rare earth metals is also very low. The fluorescence signals obtained are directly proportional to the amount of probe bound and captured on a solid phase in assays that are conveniently performed in a microtiter-plate format.Furthermore, the different emission wavelengths of the lanthanide chelates have permitted the development of multi-label assays.By using different fluorescence filters for detection, the labels can be easily distinguished from each other in an individual hybridization reaction.