Biology Reference
In-Depth Information
Northern Blotting
Total RNA was isolated using the TRIZOL reagent (BRL Life Technologies,
Rockville, MD). 20
µ
g RNA was separated by electrophoresis on denaturing
17% polyacrylamide gels, and electroblotted onto a Hybond-N+ membrane. The
membrane was UV cross-linked and hybridized with PerfectHyb plus hybrid-
ization buffer (Sigma). DNA oligonucleotides complementary to miR159 were
end-labeled using T4 polynucleotide kinase (Roche Applied Science, Penzberg,
Germany). For RNA gel blot analysis of WRKY2, 20
µ
g total RNA was separated
on 1.5% agarose-formaldehyde gels and blotted to nylon membranes. Blots were
hybridized with [
α
-32P]dATP labeled gene-specific probes. Hybridization was
performed in PerfectHyb plus hybridization buffer (Sigma) overnight at 68°C.
The membrane was then washed for 10 minutes twice with 2× SSC (1× SSC is
0.15 M NaCl and 0.015 M sodium citrate) and 1% SDS and for 10 minutes
with 0.1× SSC and 1% SDS at 68°C. Transcripts for WRKY2 were detected with
about 1 kb before stop codon of WRKY2 cDNA as probe.
Authors' Contributions
WJ carried out all experiments of WRKY2 gene, participated in the design of the
study, drafted and edit the manuscript. DY conceived of the study, participated
in the design and helped to draft and edit the manuscript. All authors read and
approved the final manuscript.
Acknowledgements
We thank the ABRC at the Ohio State University (Columbus, OH) for the Ara-
bidopsis mutants. We are grateful to Dr. Zhixiang Chen (Department of Botany
and Plant Pathology, Purdue University, West Lafayette, Indiana, USA) for Ara-
bidopsis mutants and his critical reading of the manuscript. his work was sup-
ported by the Science Foundation of the Chinese Academy of Sciences (grant no.
KSCX2-YW-N-007), and the Hundred Talents Program of the Chinese Acad-
emy of Sciences, the Natural Science Foundation of Yunnan Province (grant no.
2003C0342M), and the Ministry of Science and Technology of China (grant no.
2006AA02Z129).
