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ARS Home » Midwest Area » Urbana, Illinois » Soybean/maize Germplasm, Pathology, and Genetics Research » Research » Publications at this Location » Publication #231948

Title: A Robust Plant RNA Isolation Method for Affymetrix Genechip® Analysis and Quantitative Real-Time RT-PCR

item Clough, Steven

Submitted to: Nature Protocols
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 11/4/2008
Publication Date: 2/23/2009
Citation: Bilgin, D.D., Delucia, E.H., Clough, S.J. 2009. A Robust Plant RNA Isolation Method for Affymetrix Genechip® Analysis and Quantitative Real-Time RT-PCR. Nature Protocols. 4:333-340.

Interpretive Summary: Extracting high quality RNA in ample quantities from plant tissues is a common obstacle in plant molecular biology research. We have developed a plant RNA isolation method that is very effective for isolating high quality plant RNA with maximum yield. When compared to other common plant RNA extraction methods, we found that our protocol produced the cleanest RNA. In fact, the RNA is so clean, that one does not need to run the samples through Qiagen columns for downstream applications such as qRT-PCR or Affymetrix analysis. We believe researchers will benefit from this protocol when analyzing plant gene expression as the RNA is of high quality and yield, and it maintains small RNA molecules that may be lost in column-based purification methods.

Technical 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 maximum yield 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 results in the loss of 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. 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 hours after sampling. Additionally, the protocol described here maintains isolation of small RNA molecules, making it an ideal choice for RNA preparation prior to high-throughput sequencing methods to study gene expression.