A standard curve based method for relative real time PCR data processing
By Alexey Larionov, Andreas Krause, William Miller. BMC Bioinformatics, 2005
961 citations recorded by the source corpus.
BackgroundCurrently real time PCR is the most precise method by which to measure gene expression. The method generates a large amount of raw numerical data and processing may notably influence final results. The data processing is based either on standard curves or on PCR efficiency assessment. At the moment, the PCR efficiency approach is preferred in relative PCR whilst the standard curve is often used for absolute PCR. However, there are no barriers to employ standard curves for relative PCR. This article provides an implementation of the standard curve method and discusses its advantages and limitations in relative real time PCR.ResultsWe designed a procedure for data processing in relative real time PCR. The procedure completely avoids PCR efficiency assessment, minimizes operator involvement and provides a statistical assessment of intra-assay variation.The procedure includes the following steps. (I) Noise is filtered from raw fluorescence readings by smoothing, baseline subtraction and amplitude normalization. (II) The optimal threshold is selected automatically from regression parameters of the standard curve. (III) Crossing points (CPs) are derived directly from coordinates of points where the threshold line crosses fluorescence plots obtained after the noise filtering. (IV) The means and their variances are calculated for CPs in PCR replicas. (V) The final results are derived from the CPs' means. The CPs' variances are traced to results by the law of error propagation.A detailed description and analysis of this data processing is provided. The limitations associated with the use of parametric statistical methods and amplitude normalization are specifically analyzed and found fit to the routine laboratory practice. Different options are discussed for aggregation of data obtained from multiple reference genes.ConclusionA standard curve based procedure for PCR data processing has been compiled and validated. It illustrates that standard curve design remains a reliable and simple alternative to the PCR-efficiency based calculations in relative real time PCR.
Subfields: Genetics, Molecular Biology
View Paper: https://www.semanticscholar.org/paper/efd199777becc8e01653b8b92b202a19e9edc378
How researchers see this paper
The researchers whose trusted networks rank it highest, strongest advocate first. Every researcher gets a different order.
In Molecular Biology
Citations in the corpus
- Mixing it up: Multiple symbiont acquisition strategies as an adaptive mechanism in the coral Stylophora pistillata (2012, background): “Regardless, it is important that threshold values are set above the baseline but as low as possible within the exponential growth phase of the amplification curve (Larionov et al. 2005; Figure 4).”
- The lack of a systematic validation of reference genes: a serious pitfall undervalued in reverse transcription-polymerase chain reaction (RT-PCR) analysis in plants. (2008, methodology): “The efficiency-corrected quantification performed by RelQuant is based on relative standard curves describing the PCR efficiencies of each primer pair (Larionov et al., 2005).”
- Immunostimulatory effect of Aloe vera (Aloe barbadensis) on non-specific immune response, immune gene expression, and experimental challenge with Saprolegnia parasitica in rainbow trout (Oncorhynchus mykiss) (2019, positive): “The changes in relative mRNA expression of IL-1, IL-6, IL-8, and TNF-α were evaluated using 2 and standard curve method (Larionov et al., 2005).”
- How good is a PCR efficiency estimate: Recommendations for precise and robust qPCR efficiency assessments (2015, neutral): “O H a D M a b c d a A R R A A K R q A S D q 1 t s s s i [ r c S N r M m C h 2 Biomolecular Detection and Quantification 3 (2015) 9–16 Contents lists available at ScienceDirect Biomolecular Detection and Quantification j o ur na l ho mepage: www.elsev ier .com/ locate /bdq riginal Article ow good is ”