Biology Reference
In-Depth Information
precipitate with 90 % ethanol and 0.5 M NH
4
OAc by incuba-
tion at −80 °C overnight.
3. Wash precipitated RNA with ice-cold 85 % ethanol and dissolve
in water (approx. 500
m
sterile microfi lter, load the RNA onto a semi-preparative
reversed-phase HPLC column. Run a linear gradient increas-
ing the acetonitrile fraction from 3 to 30 % in 40 min. Optimal
gradients should be determined experimentally. Fractions con-
taining RNA product should immediately be sealed and cooled
on ice and subsequently be analyzed by MALDI-TOF or dena-
turing PAGE.
4. The correct fractions are lyophilized or again precipitated from
ethanol. Dissolve RNA in H
2
O, and determine the concentra-
tion using the UV absorption at
μ
L). After fi ltration through a 0.22-
μ
= 260 nm and the calculated
molar extinction coeffi cient (nearest neighbor) [
21
].
5. Analyze the transcribed pre-miRNA by MALDI-TOF and
15 % denaturing PAGE using the miRNA marker and low-
range ssRNA marker.
λ
3.2 Circularization
of 5
′
-Phosphorylated
ssDNA
1. 5
-Phosphorylated ssDNA templates and secondary primers
were designed according to the pre-miRNA used (
see
Note 7
and
8
).
2. For a 40
′
μ
L reaction, mix H
2
O (26
μ
L), CircLigase buffer
(4
μ
L, 10×), ATP (2
μ
L, 1 mM), MnCl
2
(2
μ
L, 50 mM), and
5
′
-phosphorylated ssDNA (4
μ
L, 10
μ
M). Then add CircLigase
L) and mix gently. Incubate the
reaction mixture at 60 °C for 4 h and then at 16 °C for 12 h.
Inactivate the enzyme by heating to 80 °C for 10 min.
3. Dilute the ligation mixture to 300
ssDNA ligase (2
μ
L, 100 U/
μ
L with Tris-HCl buffer
(10 mM, pH 8). Transfer the solution to an Amicon Ultra-0.5 mL
3K centrifuge fi lter and centrifuge (12,000 ×
g
, 20 min, RT).
Discard the fi ltrate, add another 300
μ
μ
L buffer, and centrifuge
4. Calculate the concentration by using the calculated molar
extinction coeffi cient (nearest neighbor method) [
21
] and
determining the absorption at
again. Add 30
μ
L buffer to elute the circular ssDNA template.
= 260 nm (
A
260
).
5. For quality control analyze the ligation product (approx.
1 pmol) with a 15 % denaturing PAGE and SYBR Gold stain-
ing. The circular ssDNA template should appear at higher
molecular weights relative to the linear substrate (Fig.
2
).
λ
3.3 miRNA
Maturation
1. In a 200-
M pre-miRNA stock solution
in Dicer buffer. Place the tube into a thermocycler and heat to
90 °C for 5 min. Then slowly cool to 25 °C at 2 °C/min. This
stock solution can be stored at −20 °C for up to 2 weeks (
see
Note 9
).
μ
L tube, prepare a 1
μ
