Biology Reference
In-Depth Information
2
Materials
Use all reagents of the highest quality available and certifi ed
pyrogen/DNAse/RNAse free when possible. Prepare and dilute all
solutions using ultrapure water (prepared by purifying deionized
water to attain a sensitivity of 18 M
cm at 25 °C). Diligently fol-
low all waste disposal regulations when disposing waste materials.
Ω
2.1 Ligation
of 5
1. Labeled RNA strands: 5
′
-FAM 36 mer and 3
′
-DABCYL 36
-Terminal
Partial Strands and
Beacon Purifi cation
′
- and 3
′
mer (
see
Note 1
).
2. T4 RNA ligase 1 supplied with 10× T4 RNA ligase reaction
buffer and 10 mM ATP (New England Biolabs).
3. RNase Inhibitor.
4. PCI extraction mix: Phenol/chloroform/isoamyl alcohol
25:24:1, pH 7.5-8.0.
5. 0.1 M Triethylammonium acetate (TEAA) buffer: TEAA,
pH 7.0.
2.2 Analysis of
Ligation Product
1. SYBR Gold (Invitrogen).
2. 0.3 M 2,4,6-trihydroxyacetophenone (THAP) solution, in
ethanol.
3. 0.1 M Diammonium citrate solution (pH 6.4), in water.
2.3 Preparation
of Pre-miRNA Prior
to Fluorescence
Assaying
1. Recombinant Dicer enzyme (Genlantis, San Diego, CA, USA).
2. Dicer buffer (1×): 20 mM Tris-HCl, pH 6.8, 12.5 mM NaCl,
2.5 mM MgCl
2
, 1 mM DTT (
see
Note 3
).
2.4 Fluorescence
Assay
See
Subheading
2.3
.
3
Methods
3.1 Ligation of
5
1. Ligate two partial pre-miRNA strands with FAM- and
DABCYL-modifi ed ends (Fig.
2
).
2. For a 10 nmol reaction prepare ligation mixture with fi nal con-
centration of 40
-Terminal
Partial Strands and
Beacon Purifi cation
′
- and 3
′
μ
M 5
′
-FAM 36 mer, 40
μ
M 3
′
-DABCYL 36
mer, 1× T4 Ligase buffer, 1 mM ATP, 0.8 U/μl
μ
l RNase inhibi-
l T4 RNA ligase 1. Reaction time is 18 h at 37 °C.
3. Purify RNA by PCI extraction and precipitation according to
standard procedure [
13
] followed by RP-HPLC (
see
Note 4
).
Use a gradient from 3 to 40 % 0.1 M TEAA in acetonitrile with
fl ow rate of 1 ml/min over 40 min.
tor, 0.8 U/
μ
