Biology Reference
In-Depth Information
Characterize the 3
0
end of the cDNA as described above for the 5
0
end cDNA
analysis but use the GeneRacer 3
0
primer and 3
0
nested primer together with a
forward gene-specific primer and a nested primer 5
0
to the mature miRNA.
G. Modified RACE to Map Cleavage Sites in miRNA Processing
Drosha cleavage of miRNA primary transcripts releases the hairpin precursor
miRNA, which contains 5
0
monophosphate and 3
0
hydroxyl groups, as well as
upstream and downstream products that contain 3
0
hydroxyl and 5
0
monophosphate
groups, respectively. Based on these chemical features, the products of Drosha
processing can be analyzed by modification of the RACE methods (
Bracht et al.,
2010; VanWynsberghe et al., 2011
). Below we describe methods for analyzing the 3
0
end of precursor miRNA and the 5
0
end of 3
0
Drosha cleavage products. Similar
methods can also be used to characterize the 3
0
end of the 5
0
Drosha cleavage product
as well as the 5
0
end of precursor miRNA.
1. Characterization of the pre-miRNA 3
0
End After Drosha Cleavage
Run 20
m
g of RNA, isolated as described above in Section I, on a 15% denaturing
PAGE gel alongside 10 and 25 bp ladders. Stain the gel with EtBr and cut out a band
of gel in the sample lane that corresponds to the appropriate size range. Shear gel by
spinning through a 0.5 mL tube with multiple 21-gauge needle holes. Elute RNA by
rocking sheared gel pieces in 500
m
L of 0.3 MNaCl at RT for 4 h. Transfer sample to
a Spin-X Cellulose Acetate filter and spin at top speed for 2 min. Precipitate RNA
with isopropanol, glycogen, and NaOAc, pH 5.2. Wash RNA pellet with 75% ethanol
and resuspend the RNA pellet in 26
m
L of ddH
2
O. Dephosphorylate gel extracted
RNA by incubating with 10 U of CIP in 1
NEBuffer #3 (NEB) at 37
C for 1 h.
Extract RNA as described above in the second RNA-extraction step of Section I.
After precipitation, resuspend the RNA pellet in 3
m
L of DEPC water. Ligate depho-
sphorylated RNA to a 3
0
RNA linker with a 5
0
phosphate group and 3
0
puromycin tag
by incubating RNA at 20
C overnight in a 10
m
L total reaction that contains: 1
reaction buffer (Fermentas), 1 mM ATP, 0.1 mg/mL BSA, 20 U of RNasin
(Promega), 10 U of T4 RNA ligase (Fermentas), and 5
m
M3
0
RNA linker. Extract
RNA as described above in the second RNA-extraction step of Section I. After
precipitation, resuspend RNA pellet in 20
m
L of ddH
2
O. Purify RNA by running
on a 15% denaturing PAGE gel as described above. After gel extraction, precipitate
RNAwith isopropanol, glycogen, and NaOAc, pH 5.2. Resuspend RNA in 4
m
Lof
H
2
O. Reverse transcribe by adding 1.5
m
L of 10 mM oligo complementary to the 3
0
RNA linker and 0.5
m
L of 10 mM dNTP. Incubate at 65
C for 5 min and 4
C for
1 min. Add 2
m
Lof5
buffer, 1
m
L of 0.1 MDTT, and 0.5
m
L of RNasin. Incubate at
42
C for 2 min. Add 0.5
m
L of Superscript III Reverse Transcriptase (Invitrogen)
before incubating at 42
C for 50 min and 70
C for 15 min. Add 0.5
m
L of RNase H
and incubate at 37
C for 30 min. To characterize the 3
0
end of the cleavage product,
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