Analysis of microRNA Expression and Function - Caenorhabditis Elegans: Molecular Genetics and Development - page 215

CHAPTER 8

Analysis of microRNA Expression
and Function

Priscilla M. Van Wynsberghe * , z , 1 , Shih-Peng Chan y , { , 1 ,
Frank J. Slack y , 2 and Amy E. Pasquinelli * , 2
* Department of Biology, University of California San Diego, La Jolla, California, USA
y Department of Molecular, Cellular and Developmental Biology,Yale University, NewHaven, Connecticut,
USA
z Current address: Department of Biology, Colgate University, Hamilton, NY, USA
{ Current address: Department of Microbiology, College of Medicine, National Taiwan University, Taipei,
Taiwan

Abstract
I. Introduction
II. Rationale, Methods, and Materials
A. Worm Collection and Preparation for RNA Isolation
B. RNA Isolation Method
C. Analysis of Primary miRNAs by Agarose Northern Blotting
D. Analysis of Precursor and Mature miRNAs by PAGE Northern Blotting
E. Analysis of Primary, Precursor, and Mature miRNAs by qRT-PCR
F. 5 0 and 3 0 RACE to Identify Ends of Primary Transcripts
G. Modified RACE to Map Cleavage Sites in miRNA Processing
H. Small RNA Purification
I. cDNA Library Preparation
J. Hybridization, Cluster Generation, and Sequencing
K. Computational Data Analysis
L. RIP
M. Gel Filtration Analysis of miRNPs
N. EMSA of miRNPs
O. Affinity Purification of miRNPs by Biotinylated 2 0 -O-methyl Oligonucleotides
P. miRNA Promoter - Reporter Fusion
Q. Reporter-miRNA Target 3 0 UTR Fusion
R. Transformation
III. Summary
Acknowledgments
References

1

Equal joint contribution.

2

Co-corresponding authors.