Yuji
Hiwatashi, Rumiko Kofuji
RNA
blotting allows detection of specific RNA sequences. @RNA is fractionated by agarose gel electrophoresis, followed by
transfer (blotting) to a membrane support, then hybridization with DNA or RNA
probes.@ We usually hybridize the target
RNA with a DNA probe labeled with the radioisotope 32P.@ Procedure for Hybridization, washing and
detection are the same as for DNA gel-blot analysis (Churchfs method).@ This section 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 it 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 RNA ladder and treat the same way as 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 filter with 0.22
µm mesh)
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 Na2EDTA (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
Chromotography paper Whatman 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
The
GAPDH gene is a good constitutive control (Leech et al. 1993).@ For lab use: pPpGapC contains most 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
cloned fragment by PCR using gapC5' and gapC3' primers.
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) Sakakibara et al (2003) Development.