Using real-time (Taqman) RT-PCR to measure gene expression- a core facility of the UK .

Smith RL, Painter H, Gill DR, Hyde SC

Journal of Cystic Fibrosis, 3 S1 S5

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The European Cystic Fibrosis Conference, Birmingham, 2004

Gene therapy for cystic fibrosis has been predominantly aimed at correcting the chloride ion transport defects in the lung epithelia. Mouse and sheep lung models are widely used to assess the effectiveness of a variety of gene transfer vectors and to test delivery methods to be used in the clinic (e.g. bronchoscopy, aerosolization, etc.). Testing in these animal models requires a sensitive, fast and accurate way of measuring gene transfer. Routine protein expression studies using reporter genes have proved of limited use due to the sensitivity required to detect low levels of expression that may still be therapeutic. At the John Radcliffe Hospital in Oxford, we have established a Core Testing Service using the ABI7700 Sequence Detector (TaqmanÆ) to quantify vector delivery (DNA) and gene expression (mRNA). Measurement of gene transfer using the reverse transcriptase polymerase chain reaction (RT-PCR) in real time with the TaqmanÆ can be performed in a range of pre-clinical and patient samples including blood, tissue biopsies, airway brushings and bronchial washings. Samples are collected in RNAlaterÆ stabilization solution (Ambion), transported to the core facility and the RNA extracted using the RNEasyÆ mini kit (Qiagen). A reverse transcriptase step is performed, and the resultant cDNA amplified using the TaqmanÆ instrument. An endogenous gene is analyzed simultaneously and the quantitative result normalized to the endogenous expression level. The sensitivity of the assay is 102 target RNA molecules in ???ng of RNA, with intra-assay variability typically less than 2%. RT-PCR in real time is a reliable and sensitive method to assess gene transfer efficiency and is recommended as an end-point assay for clinical assessment of gene transfe