Abstract

We developed a one-step real-time duplex reverse transcription PCR (RT-PCR) method using the LightCycler platform. This method allows simultaneous reverse transcription and real-time PCR amplification of two mRNAs of specific genes of interest (analyte genes) and mRNA of constantly transcribed genes (housekeeping genes) in a single-tube reaction. Specimen total nucleic acids were used because eukaryotic cDNA is discriminated from genomic DNA using exon-spanning primers and/or fluorescence resonance energy transfer (FRET) probes. Transcripts of murine arginase I and hypoxanthine-phosphoribosyl transferase (HPRT; housekeeping gene) or murine arginase II analyte and porphobilinogen deaminase (PBGD; housekeeping gene) were quantified in such duplex RT-PCRs. Twenty-minute reverse transcription reactions at 55 degrees C followed by 18 high-stringency step-down thermal cycles and 25 relaxed-stringency fluorescence acquisition cycles produced sensitive and accurate RT-PCR results. Fluorescent signal spillover between channels was fully compensated. A matrix of duplex PCRs at variable ratios of target standards yielded equations for factors that correct PCR-specific target ratio-dependent deviations in quantification. The one-step real-time duplex RT-PCRs reliably and accurately determined 10-10,000 copies of each target over a 100,000-fold range of target copy ratios (analyte to housekeeping mRNA = 10(-2.5)-10(2.5)) in a single assay.