5.3　 RNA gel-blot analysis

Yuji Hiwatashi, Rumiko Kofuji

Introduction

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 (Church’s 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.

Solution required

All reagents should be RNase-free.　 Use RNase-free water (ex. DEPC-treated milli-Q).

・ total RNA or poly(A)+RNA

・ 0.16-7.7Kb RNA ladder (BRL)

・ Seakem GTG agarose (FMC)

・ 20x MOPS (400 mM MOPS、100 mM CH3COONa、20 mM EDTA, adjusted pH 7.0 with NaOH, then filter with 0.22 µm mesh)

・ Formaldehyde

・ Formamide

・ 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

・ 20x SSC

・ 0.05M NaOH

Apparatus required

・ 65˚C Heat block

・ Electrophoresis apparatus

・ Hybond N+ nylon membrane (Amersham)

・ Chromotography paper Whatman 3MM

・ Paper towel

・ Incubator　

Key points

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.