Reliability of real-time reverse-transcription PCR in clinical diagnostics: gold standard or substandard?

Abstract

Molecular diagnostics is one of the major growth areas of modern medicine, with real-time PCR established as a qualitative and quantitative technology that is rapid, accurate and sensitive. The sequencing of the human genome, comprehensive genomic, mRNA and miRNA expression profiling of numerous cancer types, the ongoing identification of disease-associated polymorphisms and the expanding availability of genomic sequence information for human pathogens has opened the door to a wide range of translational applications for this technology. Consequently, novel real-time PCR assays have been developed for diagnosis and prognosis, treatment monitoring, transplant biology and pathogen detection, as well as more controversial uses such as lifestyle genotyping. However, this technology is still troubled by significant technical deficiencies. Hence its often-improper use as a clinical tool has important public health implications, most recently demonstrated through its association with the measles, mumps and rubella vaccine/autism controversy. This serves as a timely reminder of the indispensable requirement for careful experimental design, validation and analysis.

Custom Design of a GeXP Multiplexed Assay Used to Assess Expression Profiles of Inflammatory Gene Targets in Normal Colon, Polyp, and Tumor Tissue

“…22,25,26,33 Thus, it is not unexpected that the three technology platforms yield slight variations in significantly altered genes.…”

Impact of Long-Term Storage on Stability of Standard DNA for Nucleic Acid-Based Methods

“…For practical routine application, these data suggest the proximate production of standards for real-time PCR. Nevertheless, the possibility of long-term storage of DNA standards is desired, as it theoretically offers reliable data from one source and is time saving, but it is not without controversy (12,14). Schaudien et al investigated the stability of short DNA fragments (144 to 203 bp) in a short-term, rapid freeze and thaw study (19).…”

Growth and Virulence Properties of Biofilm-FormingSalmonellaentericaSerovar Typhimurium under Different Acidic Conditions

“…Over the last decades, a number of immunological and molecular based methods have been developed for rapid and specific detection of pathogens, which has a great impact on the fields of biotechnology, medicine, genetics, molecular biology, and food microbiology (26,35,41,43,49,53). Real-time reverse transcription (RT)-PCR, the most up-to-date tool to quantify gene expression, provides many advantages over conventional PCR, including a lower carryover contamination, shorter turnaround time, and faster quantitative analysis (15,16,18).…”