Journal
SEED SCIENCE RESEARCH
Volume 15, Issue 4, Pages 319-328Publisher
CAMBRIDGE UNIV PRESS
DOI: 10.1079/SSR2005220
Keywords
Arabidopsis; microRNA; Lycopersicon; small RNA; seed germination
Categories
Ask authors/readers for more resources
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 AAA(PLUS)) 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.
Authors
I am an author on this paper
Click your name to claim this paper and add it to your profile.
Reviews
Recommended
No Data Available