Fluorescent-dependent comparative C(t) method for qPCR gene expression analysis in IVF clinical pre-implantation embryonic testing

The use of quantitative PCR (qPCR) and other polymerase chain reaction (PCR)-based methods in the field of human in vitro fertilization blastocoel fluid analysis can potentially be utilized for assisting clinicians in embryo selection based on specific gene expression patterns. Since typical Comparative cycle threshold (C(t)) analysis utilizes one threshold for runs per gene target and requires an inherent control group, this method is inadequate for analysis of small stochastic systems, such as embryonic-derived fluid. We mathematically demonstrate analytical modifications upon the Comparative C(t) qPCR workflow to incorporate a variable fluorescence threshold (utilizing only the parameters defined in the Comparative C(t) method), and subsequently demonstrate the typical workflow in which this modified method can successfully quantifiably analyze embryonic blastocoel fluid qPCR analysis.