Quantitive assay for PCR product of hIL-8 mRNA with Enzyme-linked sandwich hybridization / 中国免疫学杂志

ABSTRACT

Objective:To establish a method which could quantify human IL-8 mRNA in peripheral blood monocytes.Methods:Two probe were designed aiming at amplicon of RT-PCR,of which one being capture probe adorned 5,end by ammonia and one being monitor probe adorned 3,end by bintony.So as,the capture probe could couple with the N-oxysuccinmide esters(NOS) group on the DNA-binding wells through covalent attachment.In this way,oligonucleotides were immobilized on the plate surface and arise straightly to capture target amplicon.Total RNA was extracted from peripheral blood mononuclear cell and IL-8 mRNA was amplified by using RT-PCR.Heating denatured products and denatured probe,were then added to wells and hybridization occurs between capture probe,target amplicon and detection probe.After the addition of Avidiu-peroxidase developing system,the optical density were read at 450 nm.Results:Sensitivity,detecting PCR product of 16 PCR cycling,of 5?10~3 PBMCs and of a dilute 1∶256 were its prior.Meanwhile,this method could get a specify result and a precise of 5.2%.Conclusion:Resulting in simplicity,sensitivity and specificity results,this method may be a good choice for quantify of PCR products.