DNA-free RNA isolation protocols for Arabidopsis thaliana, including seeds and siliques

Luis Oñate-Sánchez, Jesús Vicente-Carbajosa

High throughput applications of the reverse transcriptase quantitative PCR (RT-qPCR) for quantification of gene expression demand straightforward procedures to isolate and analyze a considerable number of DNA-free RNA samples. Published protocols are labour intensive, use toxic organic chemicals and need a DNase digestion once pure RNAs have been isolated. In addition, for some tissues, the amount of starting material may be limiting. The convenience of commercial kits is often prohibitive when handling large number of samples. We have established protocols to isolate DNA-free RNA from Arabidopsis thaliana tissues ready for RT-qPCR applications. Simple non-toxic buffers were used for RNA isolation from Arabidopsis tissues with the exception of seeds and siliques, which required the use of organic extractions. The protocols were designed to minimize the number of steps, labour time and the amount of starting tissue to as little as 10-20 mg without affecting RNA quality. In both protocols genomic DNA (gDNA) can be efficiently removed from RNA samples before the final alcohol precipitation step, saving extra purification steps before cDNA synthesis. The expression kinetics of previously characterized genes confirmed the robustness of the procedures. Here, we present two protocols to isolate DNA-free RNA from Arabidopsis tissues ready for RT-qPCR applications that significantly improve existing ones by reducing labour time and the use of organic extractions. Accessibility to these protocols is ensured by its simplicity and the low cost of the materials used.