Biology Reference
In-Depth Information
Chapter 9
Detection of microRNA Maturation Using Unmodifi ed
pre-microRNA and Branched Rolling Circle Amplifi cation
Saskia Neubacher and Christoph Arenz
Abstract
The ever-increasing number of different miRNAs and their association with a vast number of cellular
dysfunctions and diseases have initiated several groups to investigate miRNA maturation, which ultimately
leads to down regulation of a target messenger RNA (mRNA) and its downstream product. A rapid, con-
venient, and reliable assay to detect the Dicer-mediated miRNA-maturation step may facilitate research in
this fi eld. Here we describe the in vitro detection of the Dicer-mediated miRNA maturation step using
unmodifi ed pre-miRNA and branched rolling circle amplifi cation.
Key words
miRNA maturation assay, Enzyme assay, Rolling circle amplifi cation, Primer extension,
High-throughput screening
1
Introduction
The ever-increasing number of different microRNAs (miRNAs) and
their association with a vast number of cellular dysfunctions and dis-
eases have initiated several groups to investigate miRNA maturation,
which ultimately leads to down regulation of a target messenger
RNA (mRNA) and its downstream product [
1
-
5
]. miRNAs are
approximately 22-nucleotide (nt) double-stranded (ds) RNAs and
are generated in two consecutive enzymatic steps from the primary
miRNA (pri-miRNA) that is transcribed by RNA polymerase II.
After the pri-miRNA is cleaved in the nucleus by the endoribonuclease
Drosha, the resulting precursor miRNA (pre-miRNA) is exported
into the cytosol by exportin-5. There, the endoribonuclease Dicer
cleaves its substrate independent of its sequence and rather acts like
a molecular ruler. Dicer recognizes the end of the pre-miRNA
stem with its 5
-overhang and then cleaves two
phosphodiester bonds about 21−23 nt into the stem also creating
this signature 3
′
-OH and 2-nt 3
′
-end [
6
-
10
].
The main method to detect Dicer-mediated cleavage of pre-
miRNAs to mature miRNAs has been through northern blotting
′
- and 5
′
