Biology Reference
In-Depth Information
Crosslinker. Sandwich the membrane between dry Whatman 3MM paper and bake
in an oven at 80
C for 30 min. Incubate the membrane at 58-60
C with agitation
for at least 2 h in a sealed bag containing 25-50 mL of prehybridization solution [5
SSC, 7% SDS, 20 mM sodium phosphate, 0.1 mg/mL boiled, sheared salmon sperm
DNA, and 1
Denhardt
'
s solution (5 g of Ficoll, 5 g of polyvinylpyrrolidone, and 5 g
of BSA in 500 mL of ddH
2
O)].
Multiple methods can be used to make a radioactive probe to target the pri-miRNA
of interest. A simple, common method is based on the Prime-it II Random Primer
Labeling Kit (Stratagene), which requires only a gel-purified, 500-1500 bp PCR
product of the desired target and 80
m
Ci of alpha-
32
P dATP (6000 Ci/mmol). Add
probe to 25-50 mL of fresh prehybridization solution. Remove the original prehy-
bridization solution from the blot container before adding the fresh probe solution.
Incubate blot in probe solution at 58-60
C with agitation for at least 4 h or up to
overnight.
3. Washing, Viewing, and Stripping
Probe solution can be stored at
20
C and reused. Remove the blot from the bag
and transfer to a container with a fitted lid. Wash the blot twice at 58-60
C for
10 min in high salt wash solution (3
SSC, 10
Denhardt
'
s solution, 5% SDS, and
25 mM sodium phosphate), flipping the blot after 5 min for each wash. Wash the blot
once at 58-60
C for 10 min in low salt wash solution (0.5
SSC and 1.5% SDS).
Repeat wash in low salt wash solution if the radioactive signal on the blot is too high
or not specific. Wrap membrane in plastic wrap and expose to film or a phosophoi-
mager screen. To strip the blot, incubate membrane in boiling 0.1% SDS with
agitation. Repeat until radioactive signal is no longer detected.
D. Analysis of Precursor and Mature miRNAs by PAGE Northern Blotting
To ensure detection of pre- and mature miRNAs at least 20
m
g of total RNA,
prepared by the method described above in Section I, is recommended for each
sample.
1. Running and Transferring
To visualize both pre- and mature miRNAs, make an 11% denaturing, urea PAGE
gel (Sequagel, National Diagnostics). Pre-run PAGE gel in 0.5
Tris Borate EDTA
(TBE) buffer at 150 V for 30 min. Prepare RNA samples by mixing 20
m
g of total
RNA in a final volume of 15
m
L with an equal volume of 1
formamide loading dye
(10 mM EDTA, 80% deionized formamide, and 1 mg/mL xylene cyanol and brom-
phenol blue dye mix). Heat RNA samples at 65
C for 10 min before loading. Run gel
at 150 Vuntil the bromphenol blue dye is at the bottom of the gel,
1.5 h for 10 cm
10 cm gel size. Stain PAGE gel with EtBr to visualize rRNA bands and confirm even
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