Biology Reference
In-Depth Information
HO
O
N
H
S
O
O
COOH
O
HN CH
2
O P
O 5'-Oligo
6
O
-
O
1
O
O
O
H
O
NH
N
N
N
O
N
O
H
3
C
3'-Oligo
O
P
O
N
CH
3
O
O
-
2
OH
H
Fig.
2
Structure of 5′- and 3′-terminal ends. RNA beacon is formed by ligating
two 36 mers, one with the 5′-fl uorescein modifi cation (FAM-EX-5 linker,
1
) and
the other with the 3′-Dabcyl linker containing a 5′-phosphate (
2
). Linkers shown
are purchased from IBA (Germany) and may vary by supplier
4. Analyze by MALDI-TOF and PAGE. Successful ligation
leads to the 5
′
-FAM- and 3
′
-DABCYL-labeled pre-miRNA
beacon.
5. Extinction coeffi cients for RNA were calculated using the
nearest neighbor method adding extinction coeffi cients for
fl uorophore and quencher. Pure fractions are lyophilized and
stored in 1 mM ammonium citrate, pH 6.4.
3.2 Analysis of
Ligation Product
1. Check for adequate ligation by polyacrylamide gel electropho-
resis (PAGE). Use 20 % denaturing 8 M urea PAGE gels as
well as native PAGE gels in TBE buffer according to standard
procedure [
13
].
2. Stain the gels with SYBR Gold. On a native gel without stain-
ing the successful quenching of the ligation product can be
observed with ex = 491 nm (
see
Note 5
). After staining with
SYBR Gold the quenched pre-miRNA beacon can be visual-
ized (Fig.
3
).
3. Perform MALDI-TOF measurements to evaluate the ligation
product. Settings for RNA consist of a linear and negative ion
mode.
4. Prepare a matrix THAP/citrate in a ratio of 2:1 v/v from
0.3 M THAP in ethanol and 0.1 M diammonium citrate
(pH 6.4) in water.
