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Accurate and precise DNA quantification in the presence of different amplification efficiencies using an improved Cy0 method.

ABSTRACT: Quantitative real-time PCR represents a highly sensitive and powerful technology for the quantification of DNA. Although real-time PCR is well accepted as the gold standard in nucleic acid quantification, there is a largely unexplored area of experimental conditions that limit the application of the Ct method. As an alternative, our research team has recently proposed the Cy0 method, which can compensate for small amplification variations among the samples being compared. However, when there is a marked decrease in amplification efficiency, the Cy0 is impaired, hence determining reaction efficiency is essential to achieve a reliable quantification. The proposed improvement in Cy0 is based on the use of the kinetic parameters calculated in the curve inflection point to compensate for efficiency variations. Three experimental models were used: inhibition of primer extension, non-optimal primer annealing and a very small biological sample. In all these models, the improved Cy0 method increased quantification accuracy up to about 500% without affecting precision. Furthermore, the stability of this procedure was enhanced integrating it with the SOD method. In short, the improved Cy0 method represents a simple yet powerful approach for reliable DNA quantification even in the presence of marked efficiency variations.

SUBMITTER: Guescini M

PROVIDER: S-EPMC3704541 | biostudies-literature | 2013

REPOSITORIES: biostudies-literature

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Accurate and precise DNA quantification in the presence of different amplification efficiencies using an improved Cy0 method.

Guescini Michele M Sisti Davide D Rocchi Marco B L MB Panebianco Renato R Tibollo Pasquale P Stocchi Vilberto V

PloS one 20130708 7

Quantitative real-time PCR represents a highly sensitive and powerful technology for the quantification of DNA. Although real-time PCR is well accepted as the gold standard in nucleic acid quantification, there is a largely unexplored area of experimental conditions that limit the application of the Ct method. As an alternative, our research team has recently proposed the Cy0 method, which can compensate for small amplification variations among the samples being compared. However, when there is ...[more]

PMID: 23861909

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Project description:BackgroundSystemin has been extensively studied since it was discovered and is described as a peptidic hormone in tomato plants and other Solanaceae. Jasmonic acid and systemin are proposed to act through a positive feed-back loop with jasmonic acid, playing synergistic roles in response to both wounding and insect attack. Despite its biological relevance, most studies regarding the function of systemin in defence have been studied via PROSYSTEMIN (PROSYS) gene expression, which encodes the propeptide prosystemin that is later cleaved to systemin (SYS). Interestingly, hardly any studies have been based on quantification of the peptide.ResultsIn this study, a simple and accurate method for systemin quantification was developed to understand its impact on plant metabolism. The basal levels of systemin were found to be extremely low. To study the role of endogenous systemin on plant metabolism, systemin was quantified in a transgenic line overexpressing the PROSYS gene (PS+) and in a silenced antisense line (PS-). We evaluated the relevance of systemin in plant metabolism by analysing the metabolomic profiles of both lines compared to wildtype plants through untargeted metabolomic profiling. Compounds within the lignan biosynthesis and tyrosine metabolism pathways strongly accumulated in PS+ compared to wild-type plants and to plants from the PS- line. The exogenous treatments with SYS enhanced accumulation of lignans, which confirms the role of SYS in cell wall reinforcement. Unexpectedly, PS+ plants displayed wild-type levels of jasmonic acid (JA) but elevated accumulation of 12-oxo-phytodienoic acid (OPDA), suggesting that PS+ should not be used as an over-accumulator of JA in experimental setups.ConclusionsA simple method, requiring notably little sample manipulation to quantify the peptide SYS, is described. Previous studies were based on genetic changes. In our study, SYS accumulated at extremely low levels in wild-type tomato leaves, showed slightly higher levels in the PROSYSTEMIN-overexpressing plants and was absent in the silenced lines. These small changes have a significant impact on plant metabolism. SA and OPDA, but not JA, were higher in the PROSYS-overexpressing plants.

Dataset Information

Accurate and precise DNA quantification in the presence of different amplification efficiencies using an improved Cy0 method.

Publications

Accurate and precise DNA quantification in the presence of different amplification efficiencies using an improved Cy0 method.

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