Over the past few years high-throughput platforms for real-time quantitative PCR have become widely available. The cost of RNA extraction from a large number of samples are, however quite notable. One method that stands out with respect to free up- or downscaling of sample size and reliability is the isolation of mRNA using oligodeoxythymidylate [oligo(dT)(25)]-coated magnetic particles. In combining this magnetic separation of mRNA with real-time reverse transcription PCR (RT-PCR), we have achieved a highly reproducible, economic, and fast way of analyzing large sample numbers. One difficulty that has so far prevented the fusion of these techniques relates to accurate mRNA quantification. We present a solution to this problem that enables excellent adjustment of cDNA amounts prior to the real-time PCR. Furthermore, as the mRNA is rapidly isolated from crude plant extracts, our method is widely applicable to herbaceous plant species and various tissue types without cumbersome adjustments. Although designed and tested here for plants, we anticipate that the principles should be applicable to gene expression studies in any other organism. Lastly, due to its flexibility, the method presented here can easily be adapted to specific requirements of various users and has great potential for further automation.
|Publication status||Published - 2007|