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Fluorescent-increase kinetics of different fluorescent reporters used for qPCR depend on monitoring chemistry, targeted sequence, type of DNA input and PCR efficiency.

ABSTRACT: The analysis of quantitative PCR data usually does not take into account the fact that the increase in fluorescence depends on the monitoring chemistry, the input of ds-DNA or ss-cDNA, and the directionality of the targeting of probes or primers. The monitoring chemistries currently available can be categorized into six groups: (A) DNA-binding dyes; (B) hybridization probes; (C) hydrolysis probes; (D) LUX primers; (E) hairpin primers; and (F) the QZyme system. We have determined the kinetics of the increase in fluorescence for each of these groups with respect to the input of both ds-DNA and ss-cDNA. For the latter, we also evaluated mRNA and cDNA targeting probes or primers. This analysis revealed three situations. Hydrolysis probes and LUX primers, compared to DNA-binding dyes, do not require a correction of the observed quantification cycle. Hybridization probes and hairpin primers require a correction of -1 cycle (dubbed C-lag), while the QZyme system requires the C-lag correction and an efficiency-dependent C-shift correction. A PCR efficiency value can be derived from the relative increase in fluorescence in the exponential phase of the amplification curve for all monitoring chemistries. In case of hydrolysis probes, LUX primers and hairpin primers, however, this should be performed after cycle 12, and for the QZyme system after cycle 19, to keep the overestimation of the PCR efficiency below 0.5 %. FigureThe qPCR monitoring chemistries form six groups with distinct fluorescence kinetics. The displacement of the amplification curve depends on the chemistry, DNA input and probe-targeting. The observed shift in C_q values can be corrected and PCR efficiencies can be derived.

SUBMITTER: Ruijter JM

PROVIDER: S-EPMC4167442 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Fluorescent reporter proteins based on flavin-binding photosensors were recently developed as a new class of genetically encoded probes characterized by small size and oxygen-independent maturation of fluorescence. Flavin-based fluorescent proteins (FbFPs) address two major limitations associated with existing fluorescent reporters derived from the green fluorescent protein (GFP)-namely, the overall large size and oxygen-dependent maturation of fluorescence of GFP. However, FbFPs are at a nascent stage of development and have been utilized in only a handful of biological studies. Importantly, a full understanding of the performance and properties of FbFPs as a practical set of biological probes is lacking. In this work, we extensively characterize three FbFPs isolated from Pseudomonas putida, Bacillus subtilis, and Arabidopsis thaliana, using in vitro studies to assess probe brightness, oligomeric state, maturation time, fraction of fluorescent holoprotein, pH tolerance, redox sensitivity, and thermal stability. Furthermore, we validate FbFPs as stable molecular tags using in vivo studies by constructing a series of FbFP-based transcriptional constructs to probe promoter activity in Escherichia coli. Overall, FbFPs show key advantages as broad-spectrum biological reporters including robust pH tolerance (4-11), thermal stability (up to 60°C), and rapid maturation of fluorescence (<3 min.). In addition, the FbFP derived from Arabidopsis thaliana (iLOV) emerged as a stable and nonperturbative reporter of promoter activity in Escherichia coli. Our results demonstrate that FbFP-based reporters have the potential to address key limitations associated with the use of GFP, such as pH-sensitive fluorescence and slow kinetics of fluorescence maturation (10-40 minutes for half maximal fluorescence recovery). From this view, FbFPs represent a useful new addition to the fluorescent reporter protein palette, and our results constitute an important framework to enable researchers to implement and further engineer improved FbFP-based reporters with enhanced brightness and tighter flavin binding, which will maximize their potential benefits.

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Fluorescent-increase kinetics of different fluorescent reporters used for qPCR depend on monitoring chemistry, targeted sequence, type of DNA input and PCR efficiency.

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