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

Mathew S Box1, Vincent Coustham13, Caroline Dean1 and Joshua S Mylne12*

Author Affiliations

1 John Innes Centre, Norwich Research Park, Norwich, Norfolk, NR4 7UH, UK

2 The University of Queensland, Institute for Molecular Bioscience, St Lucia, Queensland, Australia

3 Current Address: Institut Jean-Pierre Bourgin, UMR1318 INRA-AgroParisTech, B√Ętiment 7, INRA Centre de Versailles-Grignon, Route de St-Cyr (RD10), 78026 Versailles Cedex France

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Plant Methods 2011, 7:7  doi:10.1186/1746-4811-7-7

Published: 13 March 2011

Abstract

Background

Many experiments in modern plant molecular biology require the processing of large numbers of samples for a variety of applications from mutant screens to the analysis of natural variants. A severe bottleneck to many such analyses is the acquisition of good yields of high quality RNA suitable for use in sensitive downstream applications such as real time quantitative reverse-transcription-polymerase chain reaction (real time qRT-PCR). Although several commercial kits are available for high-throughput RNA extraction in 96-well format, only one non-kit method has been described in the literature using the commercial reagent TRIZOL.

Results

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.

Conclusion

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.