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

被引:34
作者
Martin, RC [1 ]
Liu, PP [1 ]
Nonogaki, H [1 ]
机构
[1] Oregon State Univ, Dept Hort, Corvallis, OR 97331 USA
基金
美国国家科学基金会;
关键词
Arabidopsis; microRNA; Lycopersicon; small RNA; seed germination;
D O I
10.1079/SSR2005220
中图分类号
Q94 [植物学];
学科分类号
071001 ;
摘要
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.
引用
收藏
页码:319 / 328
页数:10
相关论文
共 28 条
[1]   Modulation of floral development by a gibberellin-regulated microRNA [J].
Achard, P ;
Herr, A ;
Baulcombe, DC ;
Harberd, NP .
DEVELOPMENT, 2004, 131 (14) :3357-3365
[2]   Probing microRNAs with microarrays: Tissue specificity and functional inference [J].
Babak, T ;
Zhang, W ;
Morris, Q ;
Blencowe, BJ ;
Hughes, TR .
RNA, 2004, 10 (11) :1813-1819
[3]   MicroRNA expression detected by oligonucleotide microarrays: System establishment and expression profiling in human tissues [J].
Barad, O ;
Meiri, E ;
Avniel, A ;
Aharonov, R ;
Barzilai, A ;
Bentwich, I ;
Einav, U ;
Glad, S ;
Hurban, P ;
Karov, Y ;
Lobenhofer, EK ;
Sharon, E ;
Shiboleth, YM ;
Shtutman, M ;
Bentwich, Z ;
Einat, P .
GENOME RESEARCH, 2004, 14 (12) :2486-2494
[4]   MicroRNAs: At the root of plant development? [J].
Bartel, B ;
Bartel, DP .
PLANT PHYSIOLOGY, 2003, 132 (02) :709-717
[5]   MicroRNA profiling reveals distinct signatures in B cell chronic lymphocytic leukemias [J].
Calin, GA ;
Liu, CG ;
Sevignani, C ;
Ferracin, M ;
Felli, N ;
Dumitru, CD ;
Shimizu, M ;
Cimmino, A ;
Zupo, S ;
Dono, M ;
Dell'Aquila, ML ;
Alder, H ;
Rassenti, L ;
Kipps, TJ ;
Bullrich, F ;
Negrini, M ;
Croce, CM .
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, 2004, 101 (32) :11755-11760
[6]   Role of microRNAs in plant and animal development [J].
Carrington, JC ;
Ambros, V .
SCIENCE, 2003, 301 (5631) :336-338
[7]   A gibberellin-regulated xyloglucan endotransglycosylase gene is expressed in the endosperm cap during tomato seed germination [J].
Chen, F ;
Nonogaki, H ;
Bradford, KJ .
JOURNAL OF EXPERIMENTAL BOTANY, 2002, 53 (367) :215-223
[8]   A microRNA as a translational repressor of APETALA2 in Arabidopsis flower development [J].
Chen, XM .
SCIENCE, 2004, 303 (5666) :2022-2025
[9]   REDUCTION OF BACKGROUND PROBLEMS IN NONRADIOACTIVE NORTHERN AND SOUTHERN BLOT ANALYSES ENABLES HIGHER SENSITIVITY THAN P-32 BASED HYBRIDIZATIONS [J].
ENGLERBLUM, G ;
MEIER, M ;
FRANK, J ;
MULLER, GA .
ANALYTICAL BIOCHEMISTRY, 1993, 210 (02) :235-244
[10]   Gene regulation: Ancient microRNA target sequences in plants [J].
Floyd, SK ;
Bowman, JL .
NATURE, 2004, 428 (6982) :485-486