Journal
PLANT METHODS
Volume 7, Issue -, Pages -Publisher
BMC
DOI: 10.1186/1746-4811-7-7
Keywords
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Categories
Funding
- NIH [GM083068]
- European Community [220799]
- BBSRC [BB/C517633/1]
- NIH
- ERC
- EC QLK5-CT-2001-01412 [QLK5-CT-2001-01412]
- ARC [DP0879133]
- BBSRC [BBS/E/J/000CA355] Funding Source: UKRI
- Biotechnology and Biological Sciences Research Council [BBS/E/J/000CA355, BB/C517633/1] Funding Source: researchfish
- Australian Research Council [DP0879133] Funding Source: Australian Research Council
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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.
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