Northern Blot Analysis
The protocol is divided into three sections:
1. Electrophoresis of an RNA preparation under denaturing conditions in an agarose-formaldehyde gel
2. Transfer of the RNA from the gel to a nylon or nitrocellulose membrane by upward capillary transfer
3. Hybridization analysis of the RNA sequences of interest using a labeled DNA or RNA probe.
I. Agarose/Formaldehyde Gel Electrophoresis
Prepare gel: Dissolve 0.75 g agarose in 36 ml water and cool to 60°C in a water bath. When the flask has cooled to 60°C, place in a fume hood and add 5 ml of 10xE running buffer and 9 ml formaldehyde. Pour the gel and allow it to set. Remove the comb, place the gel in the gel tank, and add sufficient 1xE running buffer to cover to a depth of ~ 1mm.
Prepare sample: Adjust the volume of each RNA sample to 6 µl with water, then add 2.5 x 6 µl freshly prepared sample denaturation mix. Mix by vortexing, microcentrifuge briefly to collect liquid, and incubate 15 min at 55°C. Cool on ice for 2 min, then add 2 µl loading dye mix.
Run gel: Run the gel in 1xE running buffer at 100 volt for 10 min, then at 65 volt for 90 min
II. Transfer of RNA from Gel to Membrane
Prepare gel for transfer: Place the gel in an RNase-free dish and rinse with changes sufficient deionized water to cover the gel for 4x20 min.
Transfer RNA from gel to membrane:
1. Fill the glass dish with enough 20xSSPE.
2. Cut 2 pieces of Whatman 1MM paper, place it on the glass plate and wet it with 20xSSPE.
3. Place the gel on the filter paper and squeeze out air bubble by rolling a glass pipet.
4. Cut four strips of plastic wrap and place over the edges of the gel.
5. Cut a piece of nylon membrane (MSI, Catalog #N00HY320F5) just large enough to cover the gel and wetted in water. Place the wetted membrane on the surface of the gel. Try to avoid getting air bubbles under the membrane.
6. Flood the surface of the membrane with 20xSSPE. Cut 5 sheets of whatman 3MM paper to the same size as membrane and place on top of the membrane.
7. Put paper towels on top of the whatman 3MM paper to a height of ~6 cm, and add a weight to hold everything in place.
8. Leave overnight.
Prepare membrane for hybridization: Remove paper towels and filter papers and recover the membrane and flattened gel. Mark in pencil the position of the wells on the membrane and ensure that the up-down and back-front orientation are recognizable. Rinse the membrane in 5xSSPE, then place it on a sheet of Whatman 3MM paper and allow to try. Place RNA-side-down on a UV transilluminator (254 nm wavelength), and irradiate for appropriate length of time.
III. Hybridization Analysis
Prepare DNA or RNA probe (>108dpm/µg):
The probe labeled with Ridiprimer DNA labelling system (Amersham LIFE SCIENCE):
1. Dilute the DNA to be labelled to a concentration of 2.5-25 ng in 45 µl of sterile water.
2. Denature the DNA sample by heating to 95-100°C for 5 min in a boiling water bath.
3. Centrifuge briefly to bring the contents to the bottom of the tube, and put on ice for 10 min.
4. Add the denatured DNA to the labelling mix and reconstitute the mix by gently flicking the tube until the blue colour is evenly distributed.
5. Add 5 µl of Redivue [32P] dCTP and mix by gently pipetting up and down. Centrifuge briefly to bring the contents to the bottom of the tube, then incubate at 37°C for 30 min.
6. The probe is purified using ProbeQuantTMG-50 micro columns (Amersham pharmacia biotech):
7. G-50 micro column preparation. Resuspend the resin in the column by vortexing, loosen the cap one-fourth turn and snap off the bottom closure. Place the column in a 1.5 ml screw-cap microcentrifuge tube for support, then pre-spin the column for 1 min at 3000 rpm in an Eppendorf model 5415C.
8. Place the column in a new 1.5 ml tube and slowly apply 50 µl of the sample to the top-center of the resin, being carful not to disturb the resin bed. Spin the column at 3000 for 2 min. The purified sample is collected in the bottom of the support tube.
Hybridization:
9. Pre-hybridization: Wet the membrane in the 5xSSPE and place it RNA-side-up in a hybridization tube and add 5 ml pre-hybridization solution, then place the tube in the hybridization oven and incubate with rotation 6 hr at 42°C for DNA probe or 60°C for RNA probe.
10. Hybridization: Double-stranded probe was denatured by heating in a water bath for 10 min at 100°C, then transfer to ice. Pipet the desired volume of probe into the hybridization tube and continue to incubate with rotation overnight at 42°C for DNA probe or 60°C for RNA probe.
Autoradiography:
11. The membrane was washed twice for 5-10 min with wash-buffer at room temperature, and twice for 15 min at 65°C.for with prewarmed (65°C) wash-buffer.
12. Remove final wash solution and rinse membrane in 5xSSPE at room temperature. Blot excess liquid and cover in UV-transparent plastic wrap. Do not allow membrane to dry out if it is to be reprobed.
13. Blot was exposed at -80°C unsing Kodak XAR film and x-ray intensifying screens.
**Reagents and Solutions**
1. 10xE: 0.2M MOPS, 0.05M NaAc and 0.005M EDTA, adjust to pH 7.0 with NaOH;
2. Sample denaturation mix (100 µl): 64.6 µl Formamide, 22.6 µl Formaldehyde and 13 µl 10xE;
3. Loading daye mix: 50% Glycerol, 0.3% Xylene cyanol, 0.3% bromophenol blue and 1mM EDTA;
4. 20xSSPE: 3M NaCl, 0.25M NaH2PO4 and 0.02M EDTA, adjust to pH 7.4;
5. Pre-hybridization solution: 2.5ml formamide, 1ml 5xP, 1.4ml H2O, 0.292g NaCl and 0.1ml sperm DNA;
6. 5xP: 0.25M Tris-HCl pH 7.5, 0.5% Sodium pyrophosphate, 1% Polyvinylpyrolidone, 1% Bovine serum albumine, 1% Ficoll and 5% SDS;
7. Wash-buffer: 1% SDS + 0.1% 20xSSPE.
Other method for RNA ELECTROPHORESIS
FORMALDEHYDE GEL:
For a 100 ml solution, add to each other:
- 1-1.5 g agarose
- 10 ml 10x MOPS buffer (not older then 2 weeks for Northern)
- 72 ml H2O
-Melt and cool down until 50°C
-Add 16 ml 37% formaldehyde
-Poor the gel (approx. 40 ml per gel)
RNA Sample Preparation:
For Northern bloting
- 5 µl RNA (max. 30 mg)
- 2 µl 10x MOPS buffer
- 10 µl formamide (de-ionised, not yellow)
- 3.5 µl formaldehyde
Incubate 10' at 70°C, put on ice.
Load with 0.02% loading dye on pre-electrophoresed gel (10')
Run 3-4 V/cm (until dye has migrated 2/3 of the gel) in 1x MOPS buffer
Stain 5' in 5 mg/ml ethidium bromide in distilled water, de-stain in water in the dark for 1 hr (overnight is preferred)
For RNA control
- 3 µl RNA
- 4 µl formamide
- 1 µl loading dye
Pipet 150 µl 1 M Guanidine thiocyanate in a casting tray.
Poor 30 ml Agarose (0.8 - 1.5 % in TBE)
with 2 µl EtBr (5mg/ml)/ 100 ml
Run 8 V/cm in 1x TBE buffer
TBE: 90 mM Tris.HCl; 90 mM NaBorate; 2 mM EDTA
Photograph gel, but prevent extended exposure. This will cause the RNA bands to fade and disappear.
10x MOPS : (for Northern not older then 2 weeks)
200 mM MOPS (3-(N-morpholino)propanesulfonic acid) 20.6 g/500ml.
80 mM Na-Acetaat.
-adjust to pH 7.0 with 2 M NaOH
10 mM EDTA.
Alternative protocol:
*Prehybridization/Hybridization Solution
0.1% SDS
50% Formamide
5x SSC
50 mM NaPO4, pH 6.8
0.1% Sodium Pyrophosphate
5x Denhardt's Solution
50 ug/ml Sheared Herring Sperm DNA
1). Air dry blot. Crosslink in Stratalinker or 30 seconds on UV Transilluminator. Wash the blot for 30 minutes in 1x SSC, 0.1% SDS at 65C.(optional)
2). Drain solution and replace with prehyb solution. Prehyb for at least 2 hours at 42C.
3). Denature the probe at 95C for 5 minutes.
4). Replace prehyb solution with fresh, preheated hyb solution containing 1 million cpm of probe per ml of hyb solution. (Average hyb is 10 million cpm total).
5). Hybridize overnight at 42C.
6). Wash # 1: Fresh prehyb solution, 30 minutes at RT.
7). Wash #2 and #3: 2x SSC, 0.1%SDS 30 minutes each at RT.
8). Wash #4: 1x SSC, 0.1% SDS for 30 minutes at RT.
9). Wash #5: 0.2x SSC, 0.1% SDS for 45 minutes at 55C.
10). Blot membrane dry with Kimwipe, cover with Saran Wrap and expose to film.
*Troubleshooting and Tips:
----Excessive Background on film: Speckled background indicates that there are unincorporated nucleotides in the probe.
----Blotchy backround indicates non-specific hybridization: This can be caused by salt still on the blot before the hyb. Be sure to wash the membrane. Can also indicate a lack of agitation during the hyb.
----No signal:
a) Probe targets intronic sequences.
b) Probe not denatured prior to hyb.
c) Hyb conditions too stringent.
d) Washing conditions too stringent.
----Hybridization of Ribosomal RNA:
a) Too much total RNA has been blotted. Do not exceed 20ug of total RNA or try to use poly A+ instead.
b) Probe is too sticky. Increase the stringency of the hyb conditions.
Smears: The RNA has degraded. Stain the gel with Ethidium Bromide or SYBR Green II to ensure the RNA is intact. Can also stain filter with Methylene Blue to ensure the RNA did not degrade during transfer process.
Stripping blots for reprobing: Add boiling 0.1% SDS, and wash for 30 minutes. Reprobe starting with step 2.
