Biology Reference
In-Depth Information
Fig. 1
Schematic representation of the BRCA-based miRNA maturation assay [
5
]
adjacent to the miRNA hybridization site is blocked (Fig.
1
, top).
The usage of SYBR Gold during the amplifi cation enables the
detection of amplicon formation in real time, and a comparison of
the resulting slopes of fl uorescence increase over time to control
measurements allows an evaluation of test compounds towards the
inhibition of Dicer-mediated miRNA maturation or of different
Dicer-containing probes [
5
].
2
Materials
All materials used for RNA handling were ordered pyrogen,
DNase, and RNase free, if possible. Glass- and metalware were
heated to 250 °C for 3 h. All surfaces were regularly treated with
an aqueous sodium hypochlorite solution (approx. 3 %) and then
rinsed with ultrapure water. Sterile gloves were used when han-
dling RNA-containing solutions. All buffers were fi ltered using a
0.22-
μ
m sterile fi lter.
2.1 In Vitro
Transcription
Components
1. T7 ssDNA template and corresponding T7 complementary
sequences for in vitro transcription of pre-miRNAs in HPLC
grade.
2. In vitro transcription kit (data optimized with T7 RiboMAX
Expression Large Scale RNA Production System, P1300,
Promega, Madison, WI, USA).
3. Triethylammoniumacetate (TEAA) buffer (0.1 M, pH 7.0)
and acetonitrile for HPLC purifi cation.
4. Reverse-phase HPLC column, e.g., Varian Polaris C18 A 5
μ
m
250 × 046 (pore size: 200 Å), heated to 55 °C.
5. miRNA Marker and low-range ssRNA marker.
6. Analytical 15 % denaturing PAGE with SYBR Gold staining.
7. Imager for visualization of SYBR Gold-stained gels.
