Biology Reference
In-Depth Information
Chapter 7
Assaying Dicer-Mediated miRNA Maturation by Means
of Fluorescent Substrates
Marlen Hesse , Brian P. Davies , and Christoph Arenz
Abstract
Assaying Dicer-mediated miRNA maturation is a valuable tool not only for validating miRNA maturation
itself but also for testing Dicer activity in cell lysate and for screening small molecules inhibiting miRNA
maturation in a high-throughput format. The classical assay for miRNA maturation relies on radioactive
labeling of a pre-miRNA and subsequent gel electrophoresis and autoradiography. Here we present a fl uo-
rescently labeled and quenched pre-miRNA beacon that can be ligated easily out of two single labeled
RNA strands. Upon Dicer cleavage of the beacon, fl uorophore and quencher are separated, which results
in an increase of fl uorescence over time. Unlike
32
P-labeled probes, our fl uorescently labeled pre-miRNA
beacon is stable for at least 5 years under storage conditions. Dicer or miRNA maturation assays can be
easily performed in a 384-well plate format, consuming less than 1 pmol of RNA beacon per reaction.
Key words
miRNA, Fluorescence assay, Nonradioactive assay, Dicer cleavage, miRNA maturation,
miRNA inhibition
1
Introduction
As abnormal expression of miRNA has been found to infl uence—
and obviously also induce—human disease, the analysis and manip-
ulation of miRNA maturation processes need easy-to-use assays for
further investigation [
1
,
2
]. Radioactive Dicer assays [
3
] or miRNA
maturation assays [
4
,
5
] are tedious because of electrophoretic
separation and autoradiography after labeling and enzyme reac-
tion. Moreover, short
32
P half-life requires repetitive labeling,
whereas appropriately stored RNA beacon is stable in our hands
for at least 5 years (
see
Note 1
).
In the special case of evaluating small-molecule inhibitors of
miRNA maturation, multiplex reactions including variation of
concentration are necessary and a homogenous assay method is
required.