LRE analysis of Real Time PCR data

LRE analysis of Real Time PCR data analyzes real-time PCR amplification curves to calculate Cy0 values for more robust and accurate nucleic acid quantification under variable PCR efficiency and in the presence of inhibitors.

Key Features:

• Cy0 Methodology: Implements the Cy0 method by fitting Richards' equation to real-time PCR data using nonlinear regression to estimate reaction parameters and compute the Cy0 value.
• Independence from efficiency assumptions: Minimizes dependency on the assumption of uniform reaction efficiency across samples by using parameter estimates from the Richards' equation fit.
• Inhibition mitigation: Reduces quantification errors caused by decreased PCR efficiency and inhibitors such as IgG that impair Ct, Cp, and sigmoidal curve fitting (SCF) methods.
• Precision and accuracy comparison: Matches Ct, Cp, and SCF in precision and accuracy under optimal amplification conditions and provides superior accuracy and precision when PCR efficiency is compromised or inhibitors are present.
• Literature basis: Based on the Cy0 approach introduced by Rutledge R.G. in the 2008 BMC Biotechnology publication.

Scientific Applications:

• Biotechnology: Sensitive DNA quantification in biotechnology workflows where variable amplification efficiency may affect results.
• Microbiology: Quantification of nucleic acids from microbial samples that may contain copurified inhibitors.
• Molecular diagnostics: Diagnostic assays requiring high precision and robustness to inhibitors and efficiency variations.

Methodology:

Richards' equation is fitted to real-time PCR data using nonlinear regression analysis to obtain parameter estimates that are used to calculate the Cy0 value.