Quantitation of gene expression by means of HPLC analysis of RT-PCR products

A method for the quantitative analysis of specific mRNA species by reverse transcription-polymerase chain reaction (RT-PCR) and subsequent detection o f products by means of ion-pair reversed-phase high-performance liquid chro matography (IP-RP-HPLC) on alkylated micropellicular polystyrene-divinylben zene particles has been developed. For RT-PCR, we used the EZrTth RNA PCR k it (Perkin Elmer). This method allows reverse transcription and amplificati on of specific target mRNA in a single reaction tube. RT-PCR products were analyzed qualitatively and quantitatively by means of IP-RP-HPLC and UV det ection at 254 nm. The enzymatic amplification combined with chromatographic separation and UV detection permitted high precision land intra assay CV < 10%), with good practicability (two pipetting steps only). A total RNA pre paration of a tissue that highly expressed the sequence of interest and tha t was stored in multiple aliquots, was diluted to give a standard curve. Th is external standard curve was used to define when samples have to be dilut ed, i.e., when the signal is in the plateau phase of amplification. The val idity of the method is demonstrated with the example of human retinoic acid receptor mRNA. (C) 1999 Elsevier Science B.V. All rights reserved.