Molecular Biology Lab 16
Northern Transfer and Hybridization Experiment #1:
Gel Electrophoresis of RNA and Capillary Transfer to Nylon
Membranes
Background:
Northern analysis is used to measure both the amount and size of RNAs in a sample. It is
one of the most basic yet important methods for analysis of gene expression. There are
several basic steps to Northern hybridization:
1.
2.
3.
4.
5.
6.
7.
Isolation of intact RNA from cells (see Lab 16-17)
Fractionation of RNA by size through a denaturing agarose gel.
Transfer of the RNA to a solid support such as nylon
Fixation of the RNA to the nylon support
Hybridization of the RNA to a probe of interest (see Lab 12-13)
Washing the blot to remove excess probe bound nonspecifically
Signal detection
Isolation of intact RNA was described in Labs 16-17, and hybridization was covered in
Labs 12-13.
Fractionation of RNA is performed by first treating RNA with formamide and then
separation in denaturing gels containing formaldehyde. Formaldehyde forms unstable
Schiff bases with imino groups on guanine residues. This inhibits base pairing by
intrastrand hybridization (hairpin loops). It is necessary to add 3-4 % formaldehyde to
both the gel and the running buffer. If you are running a minigel or performing a short
electrophoresis, you can omit formaldehyde from the gel buffer. Formaldehyde is a
teratogen (causes birth defects) and is highly toxic. It has been classified as a carcinogen
by OSHA. Please work with formaldehyde in a fume hood, if possible.
It is important to load the proper amount of RNA into each lane of the gel. Usually 10 to
20 g of total RNA is sufficient. Overloading results in large smiling bands that are thick
and rounded. One to 2 g of polyA+ RNA can also be loaded per lane.
Ribosomal RNA (consisting of 80% of all cell RNA) serves as a marker for size. The 28S
rRNA band is approximately 4.8 Kb in size and the 18S rRNA is approximately 1.9 Kb in
size. Since most mRNAs occur within this size range, the gels are run using 1.4%
agarose. However, it is also possible to purchase RNA size standards.
The gel is stained with ethidiun bromide for 30 min immediately after separation is
complete. The gel is then destained for 30 min in deionized water. RNA gels do not stain
as easily as DNA gels.
The next step is to transfer the RNA to a nylon substrate. Nylon membranes are preferred
relative to nitrocellulose due to their increased binding of nucleic acids and their
durability. If the nylon is uncharged, the capillary transfer can be performed using 10X
SSC (sodium citrate/sodium acetate). The transfer can be accomplished by upward
capillary transfer, similar to Southern blotting (see Labs 8-9).
Objectives:
The objectives of this lab are to perform gel electrophoresis to separate total cellular
RNA, and then to use capillary blotting (Northern transfer) to transfer the RNA to a nylon
membrane.
Materials:
Ethidium bromide staining solution (0.5% ethidium), funnel, and waste container
UV illuminator
Gel photography system
Microcentrifuge
Sterile deionized water
Smartspec and quartz cuvette
Ice water bath
Sample suspension buffer (formamide/10X MOPS/37% formaldehyde)
65C water bath
4 packs of paper towels
4 large glass baking dishes
Glass plates for support
Parafilm
4 scalpels or razor blades and small ruler
Dishes for staining gels
Blunt end forceps
Whatman 3M chromatography paper
20X SSC (8 liters)
6X SSC (1 liter)
Nylon membranes
500 g weight for transfer
UV cross linker
10X MOPS with 3.7% formaldehyde gel-running buffer
37% formaldehyde
1.4 % agarose
Micropipetters, sterile yellow and blue yellow tips
Sterile Eppendorf tubes
Plastic burger flipper
Large gel electrophoresis box and comb
Power packs
RNA gel loading buffer
Methods:
Resuspend the RNA in water:
1. Put on gloves before working with RNA and keep your samples of RNA in an ice
bath at all times.
2. Remove your samples of RNA from the freezer and centrifuge the eppendorf
tubes containing precipitated RNA at top speed for 20 min in the microcentrifuge.
3. After centrifugation, you should see a small white or amber colored pellet on the
bottom side of the centrifuge tube. Carefully decant the supernatant from the tube
and invert the tube on a paper towel to drain. Be careful that the RNA pellet does
not start to slide down the side of the tube.
4. Add 100-400 l of sterile deionized water to the tube (depending on size of the
pellet) and resuspend the RNA by pipetting through a yellow or blue tip. Place the
tube on ice and observe the solution carefully to see if all of the RNA has been
dissolved. This might require several min.
Measuring the RNA concentration in the sample:
5. Turn on the BioRad Smartspec and set the analysis mode to DNA/RNA. Keep all
of your RNA samples on ice during this procedure. Push the button that says
RNA.
6. Set the blank for the spectrophotometer. Add 800 l of deionized water to a
quartz cuvette and place in the spec. Push the blank button.
7. Remove 8 l of RNA suspension and add to an eppendorf tube containing 800 l
of deionized water. Mix well by shaking the tube.
8. Add the contents to the cuvette and measure the absorbance and record the value.
Remove the cuvette and decant the sample. Briefly rinse the cuvette with water
before analyzing the next sample.
9. When finished, wash the quartz cuvette with water and invert to dry on a paper
towel.
10. Calculate the concentration of RNA. Multiply the absorbance reading by 4000 (a
100 fold dilution of sample x 40 conversion factor) to get the concentration in
g/ml. Multiply this value by the sample volume (0.1-0.4 ml) to obtain the total
amount of RNA.
11. Calculate the volume of RNA solution to add for every well. Each well should
receive 10-20 g of RNA (10 g / RNA concentration in g /ml = l / 10 g).
Analysis of RNA by electrophoresis through agarose gels containing formaldehyde:
12. Cast a 1.4% agarose gel containing 1X MOPS buffer and 3.7% formaldehyde.
Add 4.2 grams of agarose, 30 ml of 10X MOPS buffer, and 240 ml of deionized
water. Microwave until the agarose is dissolved. Add 30 ml of 37% formaldehyde
solution and quickly close the top. Caution, formaldehyde is hazardous, do not
breath the fumes or get on the skin.
13. Place the bottle of agarose in a 65ºC water bath and wait several min until it is no
longer hot to touch.
14. Clean a large size gel box and gel form. Tape the form to hold the agarose and
insert the comb. Pour a gel approximately 5 mm in thickness. Quickly replace the
cover of the gel box and allow the gel to polymerize for 30 min before use. When
ready, carefully remove the comb and add gel-running buffer.
15. Hook up the leads of the gel box to the power pack. Make sure that the system
works by looking for bubbles coming from the wire electrode.
16. Set up an eppendorf tube for each lane of your gel. Place tubes on ice and add the
appropriate volume of RNA for 10-20 g / lane.
17. As soon as the RNA from all of the tubes has been sampled, add 1 ml of ethanol
and 30 l of 3M sodium acetate to the dissolved RNA. A precipitate should form
immediately. Mix the sample and return it to the -70ºC freezer for long-term
storage.
18. Add 10-15 l of sample suspension buffer to each of the tubes containing the
RNA and mix well.
19. Place the tubes in a 65ºC water bath for 10 min to denature the RNA. Immediately
cool the tubes in an ice water bath for 5 min.
20. Add 7 l of gel loading buffer to each RNA sample and mix thoroughly. Load
sample into wells of the gel and turn on the voltage to 90V. Allow the RNA to run
into the gel before you turn the voltage down to 18 V. Run the gel overnight.
21. Remove the gel and rinse briefly in tap water. Place the gel in a dish with 0.5
mg/ml ethidium bromide and stain for 30 min. After staining, rinse the gel in tap
water and destain in deionized water for 30 min. Photograph the gel for your
records. You should see 2 major bands in each lane (28S and 18S) and the bands
should be sharp with no smearing of RNA. Smearing or tails of RNA indicate
RNA degradation.
Setting up the Northern Blot:
22. Cut off any portion of the gel that is not being used. Trim the top of the gel away,
leaving a little of the wells so that you can orient the gel. Trim one or more
corners of the gel so that you can distinguish it from the gels of other groups.
23. Place the gel in 20X SSC and shake gently for 1 hour on a shaker platform
24. Prepare the nylon membrane for transfer. Measure the length and width of your
gel. Cut a piece of nylon membrane using a ruler and scalpel or sharp razor. The
size of the membrane should be about 1-2mm larger than the underlying gel. Do
not touch the nylon with your bare fingers because you will transfer oil from your
skin. Wear gloves. Try to handle the membrane by its edges using forceps.
25. Place the nylon membrane into distilled or deionized water to rehydrate (turn
darker gray). If the membranes fail to wet completely after several minutes,
discard. Allow to wet for 5 min.
26. Place 1 liter of 10X SSC in a large glass baking dish. Place a solid support (such
as an inverted gel form in the bottom of the dish to support the gel.
27. Cut pieces of 3M chromatography paper about 8 inches wide and 10 inches long.
Lay a stack of 3 to 4 chromatography paper towels directly over the plastic gel
form. Allow the ends to be submerged in the 10X SSC. Using a plastic pipette,
gently roll out any air bubbles that are present under the 3M papers (you might
have to snap the ends off of the pipette). The 3M paper forms a wick that carries
10X SSC up to the gel.
28. Place the gel directly on top of the wet 3M filter paper. Gently add some 10X
SSC to the top of the gel and roll out any air bubbles between the gel and the
underlying paper using a plastic pipette. Air bubbles will completely block
transfer of RNA and will make spots on your final hybridization experiment.
29. Place the nylon membrane over the top of the gel. Add some 10X SSC and use a
pipette to gently smooth out any air bubbles. Once the nylon has been positioned,
do not to move it again.
30. Place pieces of parafilm tightly around the edges of the gel. This serves to prevent
the paper towels from sagging and touching the wick. This would be a problem
because it would short circuit the fluid flow around the gel rather than through it.
It would cause poor transfer.
31. Place several pieces of 3M paper or thicker blot paper over the nylon membrane.
Wet the layers with 10X SSC and then roll out air bubbles with a plastic pipette.
32. Stack paper towels on top of the gel to about 4 inches in height. Place a weight of
approximately 500 grams on top of the paper towels.
33. Allow the capillary transfer to run overnight. The salt solution should carry all of
the RNA from the gel to the underside of the filter. The next day, the paper towels
should be wet from the fluid sucked out of the baking dish. Place enough 10X
SSC in the baking dish (at least 1 liter) so that it is not totally depleted.
34. After the transfer is complete, remove the paper towels down to the level of the
membrane. Carefully remove the membrane and place in 6X SSC on a shaker for
5 minutes. This washes away any agarose that might stick to the membrane.
35. After washing, place the membrane on paper towels and allow to become semi
dry (5-10 min). Place the membrane in a UV cross linker and irradiate with 1.5
J/cm2 to cross link DNA to the membrane.
36. Store the membrane at 4ºC until you are ready to make probe and hybridize.