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A robust plant RNA isolation method suitable for Affymetrix GeneChip analysis and quantitative real-time RT-PCR

Abstract

Microarray analysis and quantitative real-time RT-PCR are the major high-throughput techniques that are used to study transcript profiles. One of the major limitations in these technologies is the isolation of large quantities of highly pure RNA from plant tissues rich in complex polysaccharides, polyphenolics and waxes. Any contamination of the isolated RNA affects the downstream applications and requires extra cleaning procedures that result in a reduced RNA yield, especially the low molecular weight molecules. The protocol presented here is suitable for isolating high yield and clean total RNA from field-grown plants. Unlike current methods, such as LiCl and TRIZOL, with this new method, the isolated RNA can be used directly for Affymetrix GeneChip labeling or real-time RT-PCR without further purification. This fast and simple protocol provides ready-to-use RNA within 4–5 h after sampling. Additionally, the protocol described here maintains the isolation of small RNA molecules, making it an ideal choice for plant RNA preparation prior to high-throughput sequencing methods to study gene expression.

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Figure 1: Flow diagram to summarize the major steps of total RNA isolation with the DB protocol.
Figure 2: Nanodrop spectrophotometric measurements at A230, A260 and A280 of RNA purified using various methods.
Figure 3: The quality of the RNA isolated using various methods measured with an Agilent 2100 Bioanalyzer microfluidic electrophoresis chip.
Figure 4: Agarose (1.2%) (wt/vol) gel electrophoresis of the RNA (5 μg) isolated with various methods and from various plant species.
Figure 5: Results of Affymetrix hybridization statistics.
Figure 6: Amplification plot showing the expression level of small subunit of ribulose bisphosphate carboxylase gene (Rubisco SSII) measured by Sybergreen qPCR.

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Acknowledgements

We thank W.M. Keck Center for Comparative and Functional Genomics staff for their expertise in Affymetrix GeneChip hybridizations and Dr Metin Bilgin for a critical reading of the manuscript. This research was supported by the Office of Science (BER), U.S. Department of Energy, Grant no. DE-FG02-04ER63489 and U.S. Department of Agriculture ARS CRIS 3611-21000-018-00D.

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Correspondence to Steven J Clough.

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41596_2009_BFnprot2008249_MOESM399_ESM.pdf

Supplementary Fig. 1: Bioanalyzer electropherogram for fragmented labeled cRNA from soybean leaves. The fragment size is less than 200 nt (a) total RNA isolated with DB protocol (b) Qiagen column cleaned total RNA (PDF 152 kb)

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Bilgin, D., DeLucia, E. & Clough, S. A robust plant RNA isolation method suitable for Affymetrix GeneChip analysis and quantitative real-time RT-PCR. Nat Protoc 4, 333–340 (2009). https://doi.org/10.1038/nprot.2008.249

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