Development of a standard operating procedure (SOP) for the precise quantification of transgene expression levels in...

Variation in transgene expression levels can result from uncontrolled differences in experimental protocols. It is important to quantify and eliminate this unwanted variation as much as possible in order to attain precision in transgenic studies. Large-scale transgenic studies could, by their design, generate additional variation. The influence of different plant growth, sampling and analysis strategies in generating spurious variation in transgene expression level quantification in rice plant populations was assessed. The use of multiple independent plant phenotypic analyses (enzymatic assays in this study) was identified as the major source of spurious variation (doubling or tripling the variation). The quantification of transgene expression levels was also found to be significantly influenced by plant age, the choice of leaf sampled and leaf size. All of these factors reduced the precision of molecular genetic studies and generated artefactual results in transgenic studies. Identification of the sources of extraneous variation allowed the development of a new standard operating procedure (SOP) for rice, designed to control spurious variation. SOP allowed the influence of differences in growth period and independent phenotypic analyses to be minimized. The coefficient of variation in transgene expression levels, between and within genetically identical rice plants, was reduced to approximately 10 to 15% using SOP. Adoption of quality assurance (QA) criteria such as SOP is key to improving the reproducibility of transgenic studies.