Biology Reference
In-Depth Information
Fig. 4
Design and sequences of a BRCA system for
Drosophila melanogaster
pre-let-7/let-7 (
a
) and pre-
bantam/bantam (
b
). The mature miRNA strand used for amplifi cation is shown in
red
, and the secondary
primer is shown in
blue
8. In principle, the basic rules of standard PCR apply to the design
of the oligonucleotides for BRCA. The 5
-phosphorylated
ssDNA sequences for circular ssDNA templates include a hybrid-
ization sequence for the miRNA and for the secondary primer.
Self-hybridization and dimer formation of the ssDNA template
and the secondary primer should be avoided. The circular
ssDNA should be around 65 nt long [
12
]. Examples for the pre-
miRNA and the corresponding circular ssDNA are shown for
D. melanogaster
pre-let-7 and pre-bantam in Fig.
4
[
5
].
′
9. Pre-miRNA is best stored as a pellet after standard ethanol
precipitation (85 % ethanol in 0.5 M NH
4
OAc (pH 7.5) at
−80 °C).
10. Be sure to include all controls for the experiment: (1) pre-
miRNA with denatured Dicer and without test compound; (2)
pre-miRNA with Dicer and without test compound; and (3)
pre-miRNA with Dicer and test compound added before
BRCA, but after Dicer maturation.
11. The miRNA maturation assay can also be performed simulta-
neously for more than one pre-miRNA for example to deter-
mine selectivities of test compounds. In that case, the miRNA
maturation is performed in the presence of pre-miRNA A and
B (both at 50 nM fi nal concentrations). An aliquot is then
added to the corresponding BRCA system for pre-miRNA A
