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Simple purification of small RNAs from seeds and efficient detection of multiple microRNAs expressed in Arabidopsis thaliana and tomato (Lycopersicon esculentum) seeds

Published online by Cambridge University Press:  22 February 2007

Ruth C. Martin ,
Po-Pu Liu  and
Hiroyuki Nonogaki
Ruth C. Martin*
Affiliation:
Department of Horticulture, Oregon State University, Corvallis, OR 97331, USA
Po-Pu Liu
Affiliation:
Department of Horticulture, Oregon State University, Corvallis, OR 97331, USA
Hiroyuki Nonogaki*
Affiliation:
Department of Horticulture, Oregon State University, Corvallis, OR 97331, USA
*
†Present address: National Forage Seed Production Research Center, USDA-ARS, 3450 SW Campus Way, Corvallis, OR 97331-7102, USA.
*Correspondence: Fax: +1 541 737 3479hiro.nonogaki@oregonstate.edu
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Abstract

MicroRNAs (miRNAs) play critical roles in the development of animals and plants. Characterizing the stage- and tissue-specific expression of miRNAs that potentially regulate target transcription factor expression is becoming more important for understanding the regulatory mechanisms of critical events during plant development. A simple method for purifying small RNAs from seeds is described, as well as an efficient non-radioactive labelling system for making miRNA probes. In Arabidopsis thaliana seed extracts, low molecular-weight (LMW) RNAs (e.g. 5S rRNA, tRNA and miRNA) were separated from high molecular-weight (HMW) nucleic acids (e.g. 28S and 18S rRNA, mRNA and genomic DNA) by fractionation using isopropanol. HMW RNAs precipitated in 20% isopropanol, while most LMW RNAs remained in the supernatant. The purified LMW RNAs were used successfully for RNA gel blotting to detect miRNAs expressed in Arabidopsis and tomato (Lycopersicon esculentum) seeds. To increase the detection sensitivity of the microRNA probes, additional digoxigenin-labelled uridine triphosphates (UTPs) were incorporated into the miRNA probes by designing template oligo DNAs with three extra adenines (A) at each end of their sequence. These DNA oligomers were used to make double-stranded DNA templates for miRNA probe synthesis. This probe (termed AAAPLUS) exhibited stronger signals than normal probes. A technique was also developed to quickly screen expressed miRNAs in seeds using a miniblot system, which enabled simultaneous examination with multiple miRNA probes. This method provides a simple alternative to microRNA microarrays to identify the major miRNAs expressed in seeds.

Keywords

ArabidopsismicroRNALycopersiconsmall RNAseed germination
Type
Research Article
Information
Seed Science Research , Volume 15 , Issue 4 , December 2005 , pp. 319 - 328
Copyright
Copyright © Cambridge University Press 2005

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