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

Mathew S Box,Joshua S Mylne,Vincent Coustham,Caroline Dean

doi:10.1186/1746-4811-7-7

Abstract

BackgroundMany 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.ResultsWe 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.ConclusionThe 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.

Highlights

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
UV-spectrophotometry and microfluidic electrophoresis show that the HTP96 protocol is able to isolate good yields of highly intact RNA (40 to 100 μg per 100 mg of starting tissue), that is free from appreciable levels of contaminating proteins, phenol or salts (A260/A280 ~2.0 and A260/A230 >2.2) and is of comparable quality to RNA isolated using a standard P:C-L extraction protocol but with improved throughput
We have shown that HTP96 RNA is suitable for immediate use in reverse transcription and real time qRT-PCR

Summary

Introduction

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. 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. Downstream technologies such as real time qRT-PCR have increased in Conventional RNA isolation techniques are based on a 1.5 mL micro-centrifuge tube format (or larger) using commercially available spin/vacuum-column kits or organic solvents such as TRIZOL (Invitrogen) and phenol. A large number of protocols have been published for isolating high quality RNA only one high-throughput 96-well protocol is apparent in the published literature This uses the commercial reagent TRIZOL (Invitrogen) for 96-well format nucleic acid extraction from Arabidopsis tissues, where it was favoured for its ability to simultaneously extract both DNA and RNA in a small number of steps [2]. Several commercial kits are available for high-throughput RNA extraction in 96-well format, only one nonkit method has been described in the literature using the commercial reagent TRIZOL

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Conclusion