Protocol: A simple phenol-based method for 96-well extraction of high quality RNA from Arabidopsis

We describe an unusual phenomenon when using TRIZOL reagent with young Arabidopsis seedlings. This prompted us to develop a high-throughput RNA extraction protocol (HTP96) adapted from a well established phenol:chloroform-LiCl method (P:C-L) that is cheap, reliable and requires no specialist equipment. With this protocol 192 high quality RNA samples can be prepared in 96-well format in three hours (less than 1 minute per sample) with less than 1% loss of samples. We demonstrate that the RNA derived from this protocol is of high quality and suitable for use in real time qRT-PCR assays.The development of the HTP96 protocol has vastly increased our sample throughput, allowing us to fully exploit the large sample capacity of modern real time qRT-PCR thermocyclers, now commonplace in many labs, and develop an effective high-throughput gene expression platform. We propose that the HTP96 protocol will significantly benefit any plant scientist with the task of obtaining hundreds of high quality RNA extractions.The scale of experiments conducted in modern plant molecular biology has grown such that hundreds or thousands of plant samples need to be processed by the researcher for use in a range of downstream applications, such as quantitative trait mapping, mutant screening and the analysis of gene expression in natural accessions-a rapidly growing resource for Arabidopsis research. Real time qRT-PCR is a common downstream application in such experiments and has become a major platform for high-throughput transcript profiling [1]. A significant bottleneck for many researchers is the acquisition of sufficient quantities of high quality RNA from such a large number of samples in a time and cost-effective manner. Although downstream technologies such as real time qRT-PCR have increased in their speed and capacity, the approaches to scale up the isolation of RNA have lagged behind.Conventional RNA isolation techniques are based on a 1.5 mL micro-centrifuge tube format (or larger)