Epithelial tumour cell detection and the unsolved problems of nested RT-PCR: a new sensitive one step method without false positive results

Battaglia, Manuela; Pedrazzoli, Paolo; Palermo, Belinda; Lanza, A.; Bertolini, Francesco; Gibelli, N.; Prada, Gian Antonio Da; Zambelli, Alberto; Perotti, Cesare; Cuna, G. Robustelli della

doi:10.1038/sj.bmt.1701405

Cited by 32 publications

(18 citation statements)

References 26 publications

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“…Therefore, the dynamic range, sensitivity and specificity of the enzyme are prime considerations for a successful RT-PCR assay. Protocols using a one tube/one or two enzyme-based approach are significantly more convenient than those using two tube/two enzymebased protocols but have been reported to be less sensitive (Battaglia et al 1998). In our hands this is reflected by a shift in the threshold cycle (C t ) corresponding to one order of magnitude of initial template copy number.…”

Section: Rtmentioning

confidence: 77%

Quantification of mRNA using real-time reverse transcription PCR (RT-PCR): trends and problems

Bustin

2002

Journal of Molecular Endocrinology

2,232

1,674

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The fluorescence-based real-time reverse transcription PCR (RT-PCR) is widely used for the quantification of steady-state mRNA levels and is a critical tool for basic research, molecular medicine and biotechnology. Assays are easy to perform, capable of high throughput, and can combine high sensitivity with reliable specificity. The technology is evolving rapidly with the introduction of new enzymes, chemistries and instrumentation. However, while real-time RT-PCR addresses many of the difficulties inherent in conventional RT-PCR, it has become increasingly clear that it engenders new problems that require urgent attention. Therefore, in addition to providing a snapshot of the state-of-the-art in real-time RT-PCR, this review has an additional aim: it will describe and discuss critically some of the problems associated with interpreting results that are numerical and lend themselves to statistical analysis, yet whose accuracy is significantly affected by reagent and operator variability.

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Section: Rtmentioning

confidence: 77%

Quantification of mRNA using real-time reverse transcription PCR (RT-PCR): trends and problems

Bustin

2002

Journal of Molecular Endocrinology

2,232

1,674

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“…Real-time RT-PCR has advantages over Northern blot or conventional RT-PCR, because it requires less RNA and provides quantitative results. We chose the two-step real-time RT-PCR method over the one-step method, because it has been reported to be more sensitive than the one-step method and less prone to problems related to production of primer-dimer artifacts and contamination with genomic DNA [19,20]. Others have reported that pseudogene sequences in genomic DNA are sometimes coamplified by primers that were expected to be specific for cDNA templates [21].…”

Section: Validation Of Real-time Rt-pcr Resultsmentioning

confidence: 99%

Quantitative analysis of gender-regulated transcripts in the filarial nematode Brugia malayi by real-time RT-PCR

Rush

Tan

et al. 2004

Molecular and Biochemical Parasitology

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Improved understanding of the biology of reproduction in filarial worms may lead to identification of new targets for drugs or vaccines. Real-time RT-PCR is increasingly being adopted for RNA quantification and genetic analysis. Candidate gender-regulated genes were selected from genes identified in prior studies by differential display RT-PCR and by electronic selection of the Brugia malayi expression sequence tag (EST) database for clusters with possible gender-specific expression (four or more transcripts in male cDNA library ESTs but none in female ESTs or vice versa). Expression of candidate genes in male and female worms was compared by real-time reverse transcription PCR with sequence-specific primers. Double stranded DNA product was measured by SYBR Green I fluorescence; melting curves and agarose gel electrophoresis were used to verify the specificity of results. Relative gene expression results were normalized by parallel studies with internal control genes that were shown to be equally expressed in male and female worms (beta actin 2B, histone H3, NADH dehydroxygenase subunit 1) and calculated by the comparative C t method. Nineteen of 31 candidate genes were verified to have reproducible, gender-biased expression with fold differences between 5 and >30,000. These included several well-known genes (for example, genes encoding major sperm protein and a microfilaria sheath protein) and many novel genes. This paper reports the first large scale use of real time RT-PCR to quantitate and study gene expression in a nematode parasite. Our results represent an important step toward improved understanding of the molecular biology of reproduction in filarial nematodes.

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“…Unlike Jothikumar et al [2006], a two-step RT-PCR method was used to compare results of conventional and real-time PCR with the same cDNA and moreover it was found that the one-step method is variable intrinsically and significantly less sensitive than the two-step method. Many researchers have reported these conclusions and it was shown that experimental variation in RT-PCR is attributable mainly to the RT step [Battaglia et al, 1998;Bustin, 2002;Stahlberg et al, 2004]. Jothikumar et al [2006] described a TaqMan 1 RT-PCR assay evaluated with few HEV isolates tested previously from four major genotypes.…”

Section: Discussionmentioning

confidence: 99%

Validation of single real-time TaqMan® PCR assay for the detection and quantitation of four major genotypes of hepatitis E virus in clinical specimens

Enouf

Reis

Guthmann³

et al. 2006

J. Med. Virol.

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Since the characterization of the genome of the hepatitis E virus (HEV) in 1990, a large genetic diversity has been described. A single real-time reverse transcription (RT)-PCR assay with TaqMan 1 technology has been validated which uses only one set of primers and probe within the ORF2 HEV region (nt 5207-5292) for the detection and quantification of the four major genotypes of HEV. This assay proved to be as efficient as the conventional RT-PCR methodology for the detection of HEV in clinical samples testing positive previously. The real-time RT-PCR and conventional RT-PCR were performed comparatively on 60 pairs of sera and stools collected during a recent outbreak of hepatitis E in Darfur. The realtime RT-PCR assay was 10-to 100-fold sensitive than for conventional RT-PCR assays used in this study with a range quantitation from 1.8 Â 10 1 to 7.2 Â 10 3 RNA copies/ml in clinical samples (serum and stools).

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Epithelial tumour cell detection and the unsolved problems of nested RT-PCR: a new sensitive one step method without false positive results

Cited by 32 publications

References 26 publications

Quantification of mRNA using real-time reverse transcription PCR (RT-PCR): trends and problems

Quantification of mRNA using real-time reverse transcription PCR (RT-PCR): trends and problems

Quantitative analysis of gender-regulated transcripts in the filarial nematode Brugia malayi by real-time RT-PCR

Validation of single real-time TaqMan® PCR assay for the detection and quantitation of four major genotypes of hepatitis E virus in clinical specimens

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