Biology Reference
In-Depth Information
2.2 Ligation
Components
1. 5
-Phosphorylated ssDNA templates and secondary primers in
HPLC grade.
2. CircLigase ssDNA Ligase kit (Epicener, Madison, WI, USA).
Includes ssDNA ligase (100 U/
′
L), 10× CircLigase reaction
buffer, ATP (1 mM), MnCl
2
(50 mM).
3. 0.5 mL 3K Centrifuge fi lter.
4. Analytical 15 % denaturing PAGE with SYBR Gold staining.
5. Imager for visualization of SYBR Gold-stained gels.
μ
1. Recombinant human Dicer (1 U/
μ
L) or cell lysate containing
2.3 miRNA
Maturation
RNAse inhibitor.
2. Pre-miRNA.
3. Dicer buffer: 20 mM Tris-HCl (pH 6.8), 12.5 mM NaCl,
2.5 mM MgCl
2
, 1 mM DTT.
4. Dicer dilution buffer: 20 mM Tris-HCl (pH 6.8), 100 mM
NaCl, 1 mM MgCl
2
, 1 mM DTT, 5 mM
β
-mercaptoethanol,
10 % glycerol, 0.1 % Triton-X 100.
5. Clear 96-well plates.
6. Incubator (
see
Note 3
).
1. Circular ssDNA and secondary primer.
2. Dicer-processed pre-miRNA.
3. Bst polymerase (8 U/
2.4 BRCA
Components
μ
L; New England Biolabs), including
ThermoPol buffer.
4. Deoxynucleoside triphosphates (dNTP-mix).
5. Clear 96-well plates.
6. Real-time thermocycler (
see
Note 4
).
3
Methods
3.1 In Vitro
Transcription
1. T7 ssDNA template and corresponding T7 complementary
sequences for in vitro transcription of pre-miRNAs were
designed (
see
Note 5
).
2. Before the addition of T7 RNA polymerase, heat the T7
ssDNA template and T7 primer in transcription buffer to
90 °C and snap cool on ice to ensure correct hybridization. In
vitro transcription is essentially done according to the manu-
facturer's protocol in a 100-
µL scale. Extend the incubation
time to 2-4 h. After treatment with DNase for 30 min at 37 °C
(
see
Note 6
), dilute the RNA solution with H
2
O to a volume
of approximately 100-200
L. Then extract with same volume
phenol/chloroform/isoamyl alcohol (PCI, 25:24:1), and then
μ
