Measuring gene expression using reporter gene assays
Up to now, the methods described in this book have been concerned with estimating gene transcription rates by measuring RNA levels, either absolutely or in one cell relative to another. You will now appreciate that isolating RNA and measuring RNA levels are tricky techniques, which are prone to variability and potential error. If you simply want to ﬁnd out whether a single gene has its expression switched on or off under certain growth conditions, or in certain cells, then direct measurement of transcript levels can seem rather daunting. Is there an easier way? Well, the answer is ‘yes’, provided you are prepared to accept that the measurements you make might give a false impression concerning the absolute up-or down-regulation of transcription, but will give conﬁrmation that up-or down-regulation occurs. The answer is to use reporter genes, which encode either easily assayable enzymes, or protein epitopes against which commercially available antibodies are available for western blot analysis. Hence, the ouput of gene expression measured is not the ephemeral, difﬁcult-tolocate transcript, but the stable, easy-to-quantify reporter protein.