Comparison of the RNA-amplification based methods RT–PCR and NASBA for the detection of circulating tumour cells

Abstract

Increasingly, reverse transcriptase polymerase chain reaction (RT–PCR) is used to detect clinically significant tumour cells in blood or bone marrow. This may result in a redefinition of disease-free and clinical relapse. However, its clinical utility may be limited by lack of automation or reproducibility. Recent studies have suggested nucleic acid sequence-based amplification of target RNA may be more robust. In this study, nucleic acid sequence-based amplification was established to detect melanoma, colorectal and prostate cancer cells. Nucleic acid sequence-based amplification and RT–PCR both successfully amplified target RNA in peripheral blood samples from patients with melanoma and colorectal cancer, but only RT–PCR detected PSA in blood samples from patients with prostate cancer. There was relatively good agreement between sample replicates analyzed by RT–PCR (Kappa values of one for tyrosinase, 0.67 for CK-20 and one for PSA), but less agreement when analyzed by nucleic acid sequence-based amplification. This may limit the routine use of NASBA for the detection of clinically significant disease. In summary, RT–PCR appears at present to be the most reliable and reproducible method for the detection of low-level disease in cancer patients, although prospective studies are warranted to assess the clinical utility of different molecular diagnostic methods. British Journal of Cancer (2002) 86 , 102–109. DOI: 10.1038/sj/bjc/6600014 www.bjcancer.com © 2002 The Cancer Research Campaign

p16INK4A genetic and epigenetic profiles differ in relation to age and site in head and neck squamous cell carcinomas

“…It has been reported that HPV could be the causative agent responsible for the marked increase in at least a subset of HNSCC in young adults [37]. Of course, the mere presence of HPV DNA in HNSCC does not necessarily imply causation, and for HPV-mediated malignant transformation to occur, HPV viral E6/E7 oncogenes must be expressed [29]. To address this, we looked for both HPV DNA and mRNA in 21 of 24 of the cases.…”

“…The isothermal NASBA reaction occurs at 41°C. Thus, double-stranded DNA is not denatured and consequently is not amplified [29]. As a result, the presence of intron-exon boundaries or the inclusion of DNase digestion steps is superfluous, and contaminating HPV DNA should not affect the efficiency of the reaction or generate false-positive results.…”

Lipid Peroxidation Correlates with HIVmRNA in Serodiscordant Heterosexual HIVpartners of Nigerian Origin

“…However the distinguishing aspect of our work implies the use of the biochemical entities (MDA and mRNA) as a potent marker of HIV infection process as well as the progression of the disease using serodiscordant heterosexual partners of black origin as subjects. Measurement of HIV mRNA is usually done using the polymerase chain reaction (RT-PCR) [44]. Detection and Measurement of cell-associated RNA species using reverse transcription and the polymerase chain reaction (RT-PCR) is not now routinely done in hospitals or commercial clinical laboratories.…”

"Pelacakan Gen Env-TM Virus Penyakit Jembrana Galur Tabanan 1995 dengan Metode Nucleic Acid Sequence Based Amplificaton (DETECTION ENV-TM GENE OF JEMBRANA DISEASE VIRUS OF TABANAN 1995 STRAIN BY NUCLEIC ACID SEQUENCE BASED AMPLIFICATION METHOD)"

“…Aplikasi dan pengembangan metode NASBA telah banyak dikembangkan untuk tujuan deteksi berbagai agen patogen seperti virus, bakteri, jamur, parasit, dan juga sel tumor (Burchill et al, 2002;Loens et al, 2005). Menurut Compton (1991) teknologi NASBA dapat diaplikasikan untuk penapisan jaringan makanan/food screening, diagnostik veteriner, aquakultur dan juga ilmu forensik.…”