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ARS Home » Pacific West Area » Corvallis, Oregon » Forage Seed and Cereal Research Unit » Research » Publications at this Location » Publication #190869
Title: DEVELOPMENT OF A SCREENING SYSTEM FOR MICRORNAS IN GERMINATING ARABIDOPSIS SEEDS

Author
item Martin, Ruth
item LIU, P - OREGON STATE UNIVERSITY
item KOIZUKA, N - OREGON STATE UNIVERSITY
item NONOGAKI, HIRO - OREGON STATE UNIVERSITY

Submitted to: Meeting Abstract
Publication Type: Abstract Only
Publication Acceptance Date: 3/31/2005
Publication Date: N/A
Citation: N/A

Interpretive Summary: This abstract describes the development of methods to isolate and screen for small RNAs (using non-radioactive probes) that are important in controlling the expression of other genes during seed development.

Technical Abstract: MicroRNAs are key regulatory molecules that play critical roles in developmental processes in plants and animals. To gain a better understanding of the regulation of gene expression in seed germination, an efficient screening system was developed to examine microRNAs present during various stages of seed germination. A simple method for separating small RNAs from genomic DNA, 18S and 28S ribosomal RNA was developed. The supernatant obtained after 2 M lithium chloride precipitation was further fractionated with isopropanol to obtain a fraction enriched for small RNA. A non-radioactive detection system was modified to increase the sensitivity of the digoxigenin-labeled UTP probe. Three additional UTPs were added to each end of the probe by adding three dATPs to both ends of the DNA template. The modified probe was able to detect various levels of microRNA expression during seed germination. By using a miniblotting apparatus with multiple lanes, we were able to test an identical seed extract with multiple microRNA probes simultaneously.