A rapid procedure for the quantitation of low abundance RNAs by competitive reverse transcription-polymerase chain reaction

Grassi, Gabriele; Zentilin, Lorena; Tafuro, Sabrina; Diviacco, Silvia; Ventura, Alessandro; Falaschi, Arturo; Giacca, Mauro

doi:10.1093/nar/22.21.4547

Cited by 35 publications

(19 citation statements)

References 16 publications

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“…In fact, RT is the major source of variation in quantitative assays since the efficiency of this process varies greatly between different samples (1,3). Several studies have reported the use of RNA molecules as competitors to correct samples for variations in RT efficiency (13,(33)(34)(35). However, the instability of RNA makes it difficult to manage multiple RNA competitors that must be incorporated into single RT reactions in very precise quantities.…”

Section: Discussionmentioning

confidence: 99%

Standard-curve competitive RT-PCR quantification of myogenic regulatory factors in chicken embryos

Alvares

Mantoani

Corrente

et al. 2003

Braz J Med Biol Res

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The reverse transcription-polymerase chain reaction (RT-PCR) is the most sensitive method used to evaluate gene expression. Although many advances have been made since quantitative RT-PCR was first described, few reports deal with the mathematical bases of this technique. The aim of the present study was to develop and standardize a competitive PCR method using standard-curves to quantify transcripts of the myogenic regulatory factors MyoD, Myf-5, Myogenin and MRF4 in chicken embryos. Competitor cDNA molecules were constructed for each gene under study using deletion primers, which were designed to maintain the anchorage sites for the primers used to amplify target cDNAs. Standard-curves were prepared by co-amplification of different amounts of target cDNA with a constant amount of competitor. The content of specific mRNAs in embryo cDNAs was determined after PCR with a known amount of competitor and comparison to standard-curves. Transcripts of the housekeeping ß-actin gene were measured to normalize the results. As predicted by the model, most of the standard-curves showed a slope close to 1, while intercepts varied depending on the relative efficiency of competitor amplification. The sensitivity of the RT-PCR method permitted the detection of as few as 60 MyoD/Myf-5 molecules per reaction but approximately 600 molecules of MRF4/Myogenin mRNAS were necessary to produce a measurable signal. A coefficient of variation of 6 to 19% was estimated for the different genes analyzed (6 to 9 repetitions). The competitive RT-PCR assay described here is sensitive, precise and allows quantification of up to 9 transcripts from a single cDNA sample.

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Section: Discussionmentioning

confidence: 99%

Standard-curve competitive RT-PCR quantification of myogenic regulatory factors in chicken embryos

Alvares

Mantoani

Corrente

et al. 2003

Braz J Med Biol Res

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“…Additionally, plasmid pSPLI-II also contains two competitor segments for cellular ␤ -actin mRNA (32) and ␤ -globin DNA (31) copy number determination, using primer pairs PCO3 and PCO4, and BA1 and BA4, respectively. These competitor segments contain a perfect match to the respective target sequences, except for the addition of 20 extra bp in the middle.…”

Section: Methodsmentioning

confidence: 99%

“…After completion of the transcription reaction, the template DNA was removed by DNase I digestion followed by purification of the newly synthesized RNA by denaturing polyacrylamide gel electrophoresis and elution from the gel. An aliquot of the purified competitor RNA preparation was measured in a ␤ counter and, according to the U content of the transcript, its concentration was evaluated from the final specific activity, as already described (32,33).…”

Section: Methodsmentioning

confidence: 99%

“…In recent years, we have developed and extensively applied a competitive PCR technology for the exact quantitation of low abundance nucleic acids in clinical samples (30)(31)(32)(33)(34)(35), including HIV DNA and RNA in different clinical settings (36)(37)(38)(39)(40)(41). Competitive PCR and RT-PCR (using internal DNA and RNA competitors, respectively) couple the sensitivity of conventional PCR to the accuracy that is necessary for the exact quantification of the target molecules, irrespective of sample nucleic acid purity, primer pair efficiency and specificity, or amplification kinetics profile.…”

Section: Introductionmentioning

confidence: 99%

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Dynamics of HIV-1 mRNA expression in patients with long-term nonprogressive HIV-1 infection.

et al. 1997

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A large number of evidences indicate that progression of HIV disease is driven by an increase in viral burden. It is still unclear, however, to what extent this is contributed by the dysregulation of the molecular mechanisms governing virus gene expression at the transcriptional or posttranscriptional levels.To address this issue, several quantitative virologic parameters (including provirus transcriptional activity and splicing pattern) were analyzed in individuals with nonprogressive HIV infection and compared with those of a matched group of progressor patients. Exact quantification was achieved by a competitive PCR procedure using a multicompetitor template.Nonprogressors were characterized by striking differences in the levels of viremia, provirus copy number, and overall levels of all viral mRNA classes in peripheral blood mononuclear cells. Additionally, the transcriptional activity of the proviral DNA in these patients was mainly engaged in the production of multiprocessed transcripts, with a pattern resembling the early phases of the experimental infection. Taken

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“…Competitor was constructed similar to strategies described earlier (Grassi et al, 1994;Schanke et al, 1994;Studer et al, 1998). The competitor (external control) was designed to contain Pou-universal primer binding sequence of ~20 bp at ends encompassing a phage sequence.…”

Section: Construction Of the Poultry Dna Competitormentioning

confidence: 99%

Development and use of quantitative competitive PCR assay for detection of poultry DNA in fish meal

Farajollahi¹,

Aslaminejad²,

Nassiry³

et al. 2009

J. Anim. Feed Sci.

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In this study we reported the development and evaluation of a quantitative competitive polymerase chain reaction (QC-PCR) for detection and quantification of poultry DNA in fish meal. A universal PCR primer pair (Pou) was used to amplify a specific region 12s rRNA from poultry DNA. Specificity of the primers was evaluated with DNA from cattle and sheep. An external control DNA was constructed by 83 bp differing in length from the poultry target sequence and used for PCR reaction together with the target DNA. DNA was extracted from thirty commercial samples of fish meal and spiked with external control DNA and was co-amplified with Pou primer pair. The signal intensity was quantified by ImageJ 1.29x and were used to compare variety of poultry pollution percentage in fish meal samples. The results of QC_PCR showed that pollution percentage was in the range of 0.1 to 3% in collected samples. This study was the first report of QC_PCR application for quantifying of poultry pollution in fish meal in Iran.

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A rapid procedure for the quantitation of low abundance RNAs by competitive reverse transcription-polymerase chain reaction

Cited by 35 publications

References 16 publications

Standard-curve competitive RT-PCR quantification of myogenic regulatory factors in chicken embryos

Standard-curve competitive RT-PCR quantification of myogenic regulatory factors in chicken embryos

Dynamics of HIV-1 mRNA expression in patients with long-term nonprogressive HIV-1 infection.

Development and use of quantitative competitive PCR assay for detection of poultry DNA in fish meal

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