Abstract

The polymerase chain reaction (PCR) and reverse transcription PCR (RT-PCR) are the most ubiquitous molecular technology in use today. However, protocols have remained more or less the same for the past thirty years. For example, RNA is enzymatically converted into cDNA, which is then subjected to a PCR reaction that involves a denaturation step carried out at 95ºC followed by annealing and polymerisation steps that may be separate or combined at a range of temperatures for between 15 seconds and 1 minute. Most pipetting continues to be carried out manually, and again the original practices and recommendations have persisted. It is interesting that none of the basic tenets of this ubiquitous technology have been modified, and it begs the question whether there are improvements that could be made to the basic workflow. At the same time there have been novel applications for RT-PCR resulting from questions regarding the biological relevance of quantifying changes to mRNA expression levels. One such application is the proximity ligation assay (PLA), which extends the use of PCR to the detection and quantification of proteins. This presentation will (i) report modifications aimed at making RT-PCR more reliable and faster, (ii) highlight some of the pipetting criteria that are relevant for successful PCR (qPCR) and describe the use of the PLA for the early detection of viable pathogens.