Estimating the copy number of transgenes in transformed cotton by real-time quantitative PCR

Abstract

Transgenic cotton has been widely employed both in commercial cultivation and basic research. It is essential to determine which plants contain the transgene and in how many copies after transgenic cotton plants are generated. A TaqMan quantitative real-time polymerase chain reaction (Tq RT-PCR) method is described here to examine transgene copy number in transgenic cotton plants. The estimation of two transgene elements, the target gene of green fluorescence protein (GFP) and the selective gene of neomycin phosphotransferase II (NPTII), is used as an example to detail each step in Tq RT-PCR procedure, including endogenous reference gene selection, reference plasmid construction, primer-probe design, DNA extraction, real-time PCR, and data analysis. Comparing with traditional Southern hybridization analysis, this method can be used efficiently in screening large number of seedlings of T0 transgenic cotton at early stage of transformation process as well as in identifying transgene homozygotes in a segregation population.