A quantitative multistandard reverse transcriptase-polymerase chain reaction assay of the cystic fibrosis transmembrane conductance regulator: Its usefulness in studying efficiency of gene transfer

Procedures to quantify cystic fibrosis transmembrane conductance regulator (CFTR) mRNA levels have already been described but are not universally acce pted, and many investigators are skeptical about quantification. To be able to accurately monitor gene therapy, we developed a quantitative multistand ard RT-PCR method. This was based on the observation that the CFTR and ribo somal phosphoprotein PO (PR-PO) genes have retained important sequence homo logies between rat and human species, allowing the use of rat RNA as an int ernal standard. A mixture of rat and human RNAs is simultaneously reverse-t ranscribed in one reaction tube and amplification of CFTR leads to rat and human amplificates with identical sizes which will be discriminated by rest riction analysis. PR-PO is analyzed similarly and serves as a control of te mplate loading. RT-PCR of different amounts of RNAs gave similar CFTR/PR-PO ratios, with a coefficient variation below 10%. This technique was applied to a cell line of cystic fibrosis tracheal gland serous cells (CF-KM4) inc ubated with a recombinant adenovirus containing the CFTR cDNA. Kinetics and dose dependency of transgene expression could be accurately quantified. Th is method is precise, reproducible, and very simple and could be applied to monitor gene therapy in minute amounts of tissue such as biopsies from cys tic fibrosis patients. (C) 2000 Academic Press.