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Plant and Cell Physiology Advance Access originally published online on August 8, 2007
Plant and Cell Physiology 2007 48(9):1379-1384; doi:10.1093/pcp/pcm103
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© The Author 2007. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oxfordjournals.org

Short Communication

Ultrasensitive Determination of Absolute mRNA Amounts at Attomole Levels of Nearly Identical Plant Genes with High-Throughput Mass Spectrometry (MassARRAY)

Rust Turakulov1, Sureeporn Nontachaiyapoom2, Keith R. Mitchelson1, Peter M. Gresshoff2,* and Artem E. Men1,2

1Australian Genome Research Facility, Level 5, Gehrmann Laboratories, Research Road, University of Queensland, St Lucia, Brisbane, 4072, Australia
2Australian Research Council Centre of Excellence for Integrative Legume Research, University of Queensland, St Lucia, Brisbane, 4072, Australia

*Corresponding author: E-mail, p.gresshoff{at}uq.edu.au; Fax, +61-7-3365-3550.


  Abstract

Detection of very small amounts of RNA based on microdissection of plant tissue is essential for modern plant biology. Mass spectroscopy technology (MassARRAY) based on SequenomTM instrumentation was adapted to determine quickly and in a high-throughput fashion (by multiplexing) the absolute amounts of mRNA of closely related soybean genes. A sensitivity of 0.1 amol (10–19) was achieved, representing as few as 1,000 mRNA molecules. This methodology eliminates the use of housekeeping genes as reference standards and has multiple applications for plant functional genomics, such as the monitoring of individual expression of paralogous genes at ultra-low expression levels and/or in extremely small tissue samples.

Keywords: Allele-specific expression — Competitive PCR — Gene expression — Multiplexing — MALDI TOF

Abbreviations: GmKAPP, Glycine max kinase-associated protein phosphatase; GmNARK, Glycine max nodulation autoregulation receptor kinase; GmNFR1, Glycine max Nod factor receptor kinase; Gm26S, Glycine max 26S rRNA; MALDI TOF MS, matrix-assisted laser desorption ionization time-of-flight mass spectrometry; SNP, single nucleotide polymorphism.

(Received May 10, 2007; Accepted July 31, 2007)
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