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

Box, Mathew S., Coustham, Vincent, Dean, Caroline and Mylne, Joshua S. (2011) Protocol: A simple phenol-based method for 96-well extraction of high quality RNA from Arabidopsis. Plant Methods, 7 1: . doi:10.1186/1746-4811-7-7


Author Box, Mathew S.
Coustham, Vincent
Dean, Caroline
Mylne, Joshua S.
Title Protocol: A simple phenol-based method for 96-well extraction of high quality RNA from Arabidopsis
Journal name Plant Methods   Check publisher's open access policy
ISSN 1746-4811
Publication date 2011-03-13
Sub-type Article (original research)
DOI 10.1186/1746-4811-7-7
Open Access Status DOI
Volume 7
Issue 1
Total pages 10
Place of publication London, United Kingdom
Publisher BioMed Central
Collection year 2012
Language eng
Formatted 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.
Keyword Time Rt-Pcr
Genes
Identification
Expression
Q-Index Code C1
Q-Index Status Confirmed Code
Institutional Status UQ

Document type: Journal Article
Sub-type: Article (original research)
Collections: Official 2012 Collection
Institute for Molecular Bioscience - Publications
 
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