Comparison of DNA and RNA based real-time PCR assays for quantitative detection of chromosomal translocations

Human malignant chromosomal translocation involved immunoglobulin gene (Ig) at chromosome 14 and bcl-2 protooncogene on chromosome 18 is frequently used as a molecular marker for lymphomas. We studied the application of real-time PCR (TaqMan PCR) for quantitative detection of the human lymphocyte cells bearing chromosomal translocation. Three approaches for real-time PCR standards were compared (1) mixing cells with and without translocation before DNA extraction, (2) dilution of plasmid DNA with translocation marker into background DNA, (3) dilution of plasmid RNA with translocation marker into background RNA with a following reverse-transcriptase reaction. This study was conducted to investigate potential differences in sensitivity, resolution and variability between different real-time assays. We found the dilution of plasmid DNA with translocation fragment at background DNA to be the most accurate and sensitive method for building real-time PCR standards.