کد مقاله کد نشریه سال انتشار مقاله انگلیسی نسخه تمام متن
10529943 960835 2005 6 صفحه PDF دانلود رایگان
عنوان انگلیسی مقاله ISI
Duplex RT-PCR and chemiluminometric hybridization assay for combined screening of the mRNAs of prostate-specific antigen and prostate-specific membrane antigen in peripheral blood
موضوعات مرتبط
مهندسی و علوم پایه شیمی شیمی آنالیزی یا شیمی تجزیه
پیش نمایش صفحه اول مقاله
Duplex RT-PCR and chemiluminometric hybridization assay for combined screening of the mRNAs of prostate-specific antigen and prostate-specific membrane antigen in peripheral blood
چکیده انگلیسی
Combined screening of the mRNAs for prostate-specific antigen (PSA) and prostate-specific membrane antigen (PSMA) has been proposed as a more useful test than the separate PSA mRNA or PSMA mRNA assays in the molecular diagnosis and monitoring of prostate cancer. We have developed a simple and highly sensitive method for the simultaneous detection of PSA mRNA and PSMA mRNA using duplex RT-PCR and a chemiluminometric hybridization assay. Total RNA from peripheral blood was reverse-transcribed using oligo(dT)20 primer followed by duplex PCR in the presence of two pairs of primers specific for PSA and PSMA. Heat-denatured biotinylated PCR products were hybridized with PSA- and PSMA-specific oligonucleotide probes immobilized in microtiter wells. The hybrids were determined by using a streptavidin-alkaline phosphatase conjugate and a chemiluminogenic substrate. Using the duplex PCR, 50 copies of PSA DNA and 5 copies of PSMA DNA can be detected with a signal/background ratio of 9.7 and 22, respectively. Analysis of samples containing total RNA corresponding to 0.04-400 LNCaP cells indicated that the detectability of the proposed method is 1 cancer cell equivalent in 10 ml of peripheral blood. The overall reproducibility of the duplex assay, including reverse transcription, PCR, and hybridization assay, ranged from 3.6 to 15.5%. The method is very simple, rapid, sensitive and suitable for high-throughput screening analysis as opposed to the commonly used nested RT-PCR assays with electrophoretic detection.
ناشر
Database: Elsevier - ScienceDirect (ساینس دایرکت)
Journal: Analytica Chimica Acta - Volume 531, Issue 2, 28 February 2005, Pages 193-198
نویسندگان
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