Application of real-time PCR to assess transgene copy numbers in GM plants and to investigate plant gene expression

Abstract

Quantitative real-time polymerase chain reaction (qPCR) is a powerful method to compare specific DNA levels across different sample populations. In combination with reverse transcription (RT) it is the most common method for either characterising or confirming gene expression patterns. This is due to the high sensitivity, specificity, accuracy and reproducibility of the method. Quantitative DNA and RNA analysis needs an appropriate control gene for accurate normalisation of data. For gene expression analysis, an ideal endogenous control gene, also called a reference or housekeeping gene, is one that is stably expressed within the samples to be compared, regardless of tissue differences, experimental conditions or treatments. Different genes had been qPCR analysed in this study with different objectives. On the one hand, the objective was to determine the transgene copy number in genetically modified rice where only one copy of the inserted transgene was desirable; and on the other hand, RT-qPCR had been applied for studies of gene expression in Prunus perisca subjected to different treatments and conditions to induce genes related to plant defence mechanisms