Quantitative determniation of CK19-mRNA

Objective To establish a bioluminescence-based immunoassay for the quantitation of CK-19mRNA.Methods To apply aequor in-based bioluminescence immunoassay for quantitating RT-PCR products of CK19-mRNA in cancer cells and in the peripheral blood samples from human volunteers.CK19-mRNA was amplified by RT-PCR with primers labeled with biotin and then hybr idized to dig oxigenin-conjugated with oligonucleotide probe.The duplex was then captured onto a streptavidin-coated microtiter plate where it reacted with digoxigenin-specific antibody conjugated with the photoprotein aequorin.The bioluminescence reaction was triggered by adding calcium ions.The amount of specific DNA was quantitated by detecting the intensity of blue light at 469 nm.Results The linear range reached 105 ,the regression coefficient was r=0.98 0.99.Meanwhile,the technique can detect as low as 22 amol/L of amplified CK19-mRNA products.In reproducibility detection,the coefficient of variation among different batches was smaller than 7%,and the coefficient of variation within the batch was smaller than 6%.Conclusion The technique showed high sensitivity,specificity and can be practically used for the detection of CK19-mRNA in peripheral blood samples.