Multiparametric duplex real-time nucleic acid sequence-based amplification assay for mRNA profiling

Nucleic acid sequence-based amplification (NASBA) is a sensitive isothermal transcription-based amplification method. We have dei,eloped real-time NASBA assays to detect mRNA coding for the estrogen receptor alpha (ESRI) and the progesterone receptor (PGR) in breast tumors by means of duplex reactions using cyclophilin B (PPIB) as the normalizing gene. Both the ESRI/PPIB and PGR/PPIB breast tumors by means a of duplex NASBA assays are highly sensitive, specific, and reproducible. Quantification is determined using external standard calibration curves and the ratio between the number of target and housekeeping gene mRNA copies. Amplification of the target gene in the duplex NASBA assay was disrupted when this latter was mixed with a large amount of the housekeeping PPIB gene, suggesting that it is preferable for the normalizing gene chosen to have an expression level comparable to the target gene. Sensitivity, and robustness of the duplex NASBA assays are assessed in breast cancer cell lines. Such a rapid and easy-to-use multiparametric duplex real-time NASBA assay could also advantageously be set up for other mRNA profiling applications.