Quantitation of RT-PCR amplified cytokine mRNA by aequorin-based bioluminescence immunoassay.
We described here a bioluminescence-based immunoassay for the quantitation of RT-PCR amplified cytokine mRNA. This technique uses a standard RT-PCR procedure, with the following modifications. The forward primer in the PCR reaction is labeled with a 5' biotin molecule. Following PCR, a digoxigenin-conjugated oligonucleotide probe is hybridized to the target biotin-labeled DNA template. The hybridized duplex is captured onto a streptavidin-coated microtiter plate. The bound product is quantitated by adding digoxigenin-specific antibodies conjugated with the photoprotein aequorin. The amount of specific DNA captured onto the plate is quantitated by triggering the bioluminescence reaction through the addition of calcium ions. This technique detected as low as 40 amol of amplified cytokine products, or 500 copies of templates when 27 PCR cycles were used. The high sensitivity of this technique enables the quantitation of target DNA during the exponential phase of the PCR reaction. The aequorin-bioluminescence assay is an alterative non-radioactive method for the quantitation of PCR products.
Xiao
L
L
Centers for Disease Control and Prevention, U.S. Department of Health and Human Services, Atlanta, GA 30333, USA.
Yang
C
C
Nelson
C O
CO
Holloway
B P
BP
Udhayakumar
V
V
Lal
A A
AA
D000331:Aequorin; D000818:Animals; D000937:Antigen-Antibody Reactions; D016207:Cytokines / Q000235:genetics*; D004076:Digoxigenin; D015870:Gene Expression; D008163:Luminescent Measurements; D051379:Mice; D016133:Polymerase Chain Reaction / Q000379:methods*; D012333:RNA, Messenger / Q000032:analysis*
D016207:Cytokines; D012333:RNA, Messenger; D000331:Aequorin; D004076:Digoxigenin