Yuji
Hiwatashi, Rumiko Kofuji
RNA blotting
allows detection of specific RNA sequences. @RNA is fractionated by agarose gel electorphoresis, followed by
transfer (blotting) to a membrane support, then hybridized with DNA or RNA
probes.@ We usually hybridize the target
RNA with DNA probe labeled with radioisotope 32P.@ Procedure for Hybridization, washing and detection are the same
way as DNA gel-blot analysis (Churchfs method).@ This part describes how to run the RNA on a denaturing gel and
how to transfer the RNA from the gel to a solid nylon membrane.
1.
Membrane preparation
Procedure
1.
Boil 1.5 g of Seakem GTG agarose in 135 ml of RNase-free water and
let cool a little.@
2.
Add 15 ml of 20x MOPS and 15 ml of 37% formaldehyde (formalin) and
mix well.
3.
Pour into gel tray (15 x 13.5 cm) when temperature of the gel
solution is below 50˚C.
4.
Add 4 vol. of RNA sample buffer into 1 vol. (10 µg) of total
RNA solution and mix by pipetting.@ For
the ladder lanes, use 2 µg of 1 kb ladder and treat the same way as with
the samples.
5.
Heat for 10 min at 65˚C, then immediately chill on ice for 5
min. @Dilute 50 ml 20X MOPS to one liter
for running buffer.
6.
Apply each sample to the gel and run between 70-85 V for about 3
hr or to a predetermined length.
7.
Stain the gel with 5 µg/ml EtBr solution for 15 min and wash
the gel with RNase-free water for 15 min.@
Omit this step when you run more than 1 µg of poly(A)+RNA.
8.
Transfer RNA to Hybond N+ membrane with 20 x SSC by the downward
method (Chomczynski, 1992).
9.
Put the membrane on chromatography paper soaked with 0.05 M NaOH
and fix for 5 min.@ Then, rinse the membrane
with 2 x SSC.
10.
Dry up the membrane at 80˚C for 1 h.@ Store it at –30˚C unless you
immediately perform hybridization.
All reagents
should be RNase-free.@ Use RNase-free
water (ex. DEPC-treated milli-Q).
E total RNA or poly(A)+RNA
E 0.16-7.7Kb RNA ladder (BRL)
E Seakem GTG agarose (FMC)
E 20x MOPS (400 mM MOPSA100 mM CH3COONaA20
mM EDTA, adjusted pH 7.0 with NaOH, then filtrate with 0.22 µM))
E Formaldehyde
E Formamide
E RNA
sample buffer (Store at -20˚C).
Formaldehyde |
1.6 ml |
Formamide |
5.0 ml |
20x MOPS |
0.5 ml |
Glycerol
dye |
1.6 ml |
Total |
8.7 ml |
@Glycerol dye
1 mg/ml Bromophenol
blue |
1 ml |
1 mg/ml Xylene
ceanol |
1 ml |
0.5 M EDTA
(pH 8.0) |
0.02 ml |
glycerol |
5 ml |
H2O |
2.98 ml |
E 20x SSC
E 0.05M NaOH
E 65˚C
Heat block
E Electrophoresis
apparatus
E Hybond N+ nylon membrane (Amersham)
E Chromotograph paper Whatmann 3MM
E Paper towel
E Incubator@
1.
For EtBr staining, you can add 0.5 µl of 1 mg/ml EtBr to
each sample directly.@ In this case,
omit the a EtBr staining step of the gel.
2.
Positive control
GAPDH gene
is the best constitutive control at (Leech et al. 1993).@ For lab use: pPpGapC contains most part of
the GAPDH cDNA in pGEM3Z vector (pPpGapC), whose PCR amplified fragment is
useful as a probe.@
pPpGapC
plasmid: the following fragment of GAPDH gene containing part of ORF and 3' UTR
was cloned into SmaI site of pGEM3Z. The direction of the fragment is the same
as lacZ on the vector.
Amplify
inserted fragment using gapC5' and gapC3' primers by PCR.
gapC5': GAG
ATA GGA GCA TCT GTA CCG CTT GTG C
gapC3': CAT
GGT GGG ATC GGC TAA GAT CAA GGT C
References
(1)
Chomczynski (1992) Anal. Biochem. 201: 134-139.
(2) Leech et
al. (1993) Plant J. 3: 51-61.
(3)
Sakakibra et al (2003) Development.