Quantitation of IL-1 beta mRNA by a combined method of RT-PCR and an ELISA based on ion-sensitive field effect transistor.
A method for quantitative RT-PCR using ELISA detection was developed and applied to the quantitation of IL-1 beta mRNA in clinical samples. To compensate for the 'tube effect' of RT-PCR, a synthetic RNA, pRSET RNA, was added to sample solutions as an internal standard and co-amplified with IL-1 beta mRNA. Sense primers for IL-1 beta and pRSET were labeled with digoxigenin and FITC, respectively, while anti-sense primers for both were labeled with biotin. Double-stranded PCR products were captured by two solid-phase pipettetips. One was coated with an anti-digoxigenin antibody and another with an anti-FITC antibody, and sandwiched by avidin-urease, and their activities were measured by coupling them with a pH-FET in a pH-measuring cell containing urea solution. The ratio of the signal intensity for IL-1 beta to that for pRSET was used to quantify the concentration of IL-1 beta mRNA. A calibration curve was obtained by using a known amount of AW 109 RNA as an external standard of IL-1 beta RNA. It was found that 10(2)-10(6) copies of IL-1 beta mRNA were measurable by the present method. Expression levels of IL-1 beta mRNA in clinical samples, such as monocytes of peripheral blood or synovial cells from patients with RA or OA, were determined.
Tsuruta
H
H
Biosensor Research Laboratories Co., Ltd., Kurashiki, Japan.
Matsui
S
S
Oka
K
K
Namba
T
T
Shinngu
M
M
Nakamura
M
M
D001483:Base Sequence; D002138:Calibration; D004797:Enzyme-Linked Immunosorbent Assay / Q000295:instrumentation* / Q000379:methods; D006801:Humans; D007375:Interleukin-1 / Q000097:blood*; D008969:Molecular Sequence Data; D009000:Monocytes / Q000276:immunology; D016133:Polymerase Chain Reaction / Q000379:methods*; D012333:RNA, Messenger / Q000097:blood*; D012015:Reference Standards; D014173:Transistors, Electronic
D007375:Interleukin-1; D012333:RNA, Messenger