A method for the rapid construction of cRNA standard curves in quantitative real-time reverse transcription polymerase chain reaction

Abstract

Quantification of nucleic acids, especially of mRNA, is increasingly important in biomedical research. The recently developed quantitative real-time polymerase chain reaction (PCR) - a highly sensitive technology for the rapid, accurate and reproducible quantification of gene expression - offers major advantages over conventional quantitative PCR. Transcript quantification is performed in the exponential phase of the PCR reaction through extrapolation of fluorescence signals from a standard calibration curve which represents the initial copy number for a given fluorescence signal. We have developed a method for gene transcript quantification which is based on a LightCycler - assisted real-time PCR in combination with a simple and rapid approach for the construction of external cRNA standards with identical gene sequences as the target gene. Synthesis of cRNAs was performed by in vitro transcription with T7 RNA polymerase followed by reverse transcription and real-time PCR. We applied this approach for transcript quantification of eukaryotic initiation factor 3 p110 (EIF3S8) mRNA in normal testicular tissue. We also present a rapid and simple strategy for the construction of cRNA standards for use in real-time PCR.