Northern blotting - gel preparation and treatment

RNA is separated under denaturing conditions; the principal

systems currently in use are the glyoxal/dimethylsulphoxide and

the formaldehyde/formamide procedures. This protocol restricts

itself to the latter. Successful Northern analysis depends on the

quality of the reagents used as well as having pure un-degraded

RNA samples.

Avoid any contamination with RNases, use sterile disposable

plastics wherever possible. Glassware may be treated by baking at

180°C overnight or incubating in 0.2%(v/v) diethylpyrocarbonate

(DEPC) followed by autoclaving or baking. Some plastics are also

suitable for DEPC treatment.

Formaldehyde/formamide protocol

Prepare the MOPS/formaldehyde gel as follows: Preheat

17.5ml of formaldehyde and 30ml 10x MOPS buffer at 55°C.

Dissolve 3 - 4.5g of agarose in 250ml of nuclease free water.

Cool to 55°C. Add the 10x MOPS buffer and formaldehyde.

Cast the gel in an appropriate enclosure and allow the gel to

set.

Prepare the RNA sample(s), using the table below:

	Volume (ml)	Final Conc.
RNA	V
formaldehyde	5.5	2.2 M
formamide	15	50%
10X MOPS buffer	1.5	0.5X
Water	8-V
Total	30

Place the sample(s) at 55°C for 15 minutes to denature. After

denaturation add 3ml of 10x nucleic acid dye loading buffer. Mix

and load onto the agarose gel.

Separate the RNA samples using lx MOPS buffer as the

electrophoresis buffer.

Following electrophoresis, if appropriate, visualize the RNA

within the gel with UV light and photograph.

Place the gel in a suitable tray or dish and cover with distilled

water. Incubate the gel with gentle agitation for 15 minutes.

Discard the water and replace with sterile 10x SSC. Agitate for

15 minutes. Repeat this step once more.

Set up the capillary blot as described.

Notes

The agarose gel is 0. 7M with respect to formaldehyde and lx

with respect to the MOPS buffer This formulation can be scaled

up or down as appropriate for the size of gel required.

SybrGreenTM or Ethidium bromide (0.01µg/ml) may be included

in the gel for visualisation. RNA does not stain as well as the

same amount of DNA with ethidium bromide. Excessive

amounts of ethidium bromide will also inhibit RNA transfer.

Other staining procedures post electrophoresis for example

ethidium bromide or acridine orange, or methods which stain

the blot, for example methylene blue may also be used.

Sample must be deproteinised. Samples may be stored at

-20°C for short periods.

SybrGreen is recommended for visualisation. When ethidium

bromide is used for visualisation the addition of ethidium

bromide to the electrophoresis buffer (0.01µg/ml) improves

results. Nucleic acid loading buffer must be prepared using

RNase free reagents/solutions.

The integrity of the RNA may be assessed by the absence of

smearing and the fluorescent signal, the ratio of 28S to 18S

RNA should be 2:1.

10x SSC or 20x SCC can be used as the transfer buffer.

Glyoxal protocol

Prepare the MOPS gel as follows: Preheat 30ml 10x MOPS

buffer at 55°C. Dissolve 3 - 4.5g of agarose in 270ml of

nuclease free water. Cool to 55°C. Add the 10x MOPS buffer.

Cast the gel in an appropriate enclosure and allow the gel to

set.

Prepare the RNA sample(s), using the table below:

	Volume (µl)	final concentration
RNA	V
DM	15	50%
6 M Glyoxal (deionized)	5.4	1M
10x MOPS buffer	3	1X
Water	8-V
TOTAL	30

Place the sample(s) at 50°C for 60 minutes to denature. After

denaturation add 3µl of 10x nucleic acid dye loading buffer. Mix

and load onto the agarose gel.

Separate the RNA samples using lx MOPS buffer as the

electrophoresis buffer.

Following electrophoresis, if appropriate, visualise the RNA

within the gel with UV light and photograph.

Set up the capillary blot as described using a neutral transfer

buffer.

Notes

Glyoxal oxidizes very rapidly. Stock solutions 40%(w/v) or 6M

must be deionized to neutral pH before use. Small aliquots can

then be stored at -20°C in tightly capped tubes. Once thawed

use only once.

Glyoxylated RNA should be run more slowly than formaldehyde

gel to prevent formation of pH gradients.

Glyoxal gels may be electrophoresed in 1x MOPS thereby

eliminating the need to recirculate the buffer.

Glyoxal may interact with ethidium bromide altering the dyes

spectral properties.

No pre treatment of the gel is required. Glyoxal is removed from

the RNA during prehybridization or any post fixation washes of

the blot.

Hybridization in bags and boxes

Protocol

Prepare the hybridization buffer, for example

Denhardt’s Buffer

Modified Church Buffer

5x SSC

0.5M phosphate buffer, pH 7.2

5x Denhardt's solution

7% (w/v) SDS

0.5% (w/v) SDS

10mM EDTA

Ensure the SDS is in solution before use. Gentle

heating may be necessary

Combine all the components, make up to the required volume.

Prepare the radiolabelled probe using the appropriate

procedure

Preheat the required volume of hybridization buffer to the

appropriate temperature.

Pre-wet the blot in a suitable buffer for example 5x SSC or 0.5M

phosphate buffer. Place the blot(s) in the hybridization buffer.

125µl of hybridization buffer per cm2 is a suitable volume.

Prehybridize for at least 30 minutes with constant agitation, at

the desired hybridization temperature (see Note 7).

When using labelled double stranded probes, pipette the

required amount into a clean microcentrifuge tube. If the volume

is less than 2µl, make up to this volume with water or TE buffer.

Denature the probe by boiling for 5 minutes and snap cool on

ice. Briefly centrifuge to draw the contents to the bottom of the

tube

Add the probe to the pre-hybridization buffer.

Hybridize overnight with gentle agitation at the required

temperature.

Prepare the stringency wash solutions. The wash solution

should be used in excess, at least 1-5 ml/cm2 of membrane.

Low stringency wash;

2x SSC, 0.1% (w/v) SDS

Medium stringency wash:

lx SSC, 0.1% (w/v) SDS

High stringency wash:

0.lx SSC, 0.1% (w/v) SDS

After the hybridization, wash the blots by incubating twice, 5

minutes each, in 2x SSC, 0.1% SDS, followed by lx SSC,

0.1% SDS for 15 minutes, and finally 0.lx SSC, 0.1% SDS for

2 x 10 minutes, at the hybridization temperature.

10)

Remove the blot from the last stringency wash, drain, wrap in

SaranWrap and expose to X-ray film, for example Hyperfilm

MP. Keep the blot moist if it is to be reprobed. If reprobing is

desired, it may be more suitable to seal the blot in a plastic

bag.

Notes

There are a wide variety of hybridization buffers used by

researchers. This Denhardt's based buffer is that used in the

quality control of all Hybond nylon membranes. A reduced

concentration of SDS has been found to give elevated

backgrounds following hybridization. The Denhardt’s

hybridization buffer may be stored at -20°C if required.

This modification of the Church and Gilbert buffer is routinely

used at Amersham Pharmacia Biotech. It has been shown to

be suitable for Southerns, Northerns, dot blots and library

screening applications. The hybridization buffer may be stored

at room temperature. Ensure the SDS is fully dissolved before

use. This may be achieved with gentle heating.

For radioactive applications use a probe concentration of 0.5 -

2 x 106 incorporated counts per ml of hybridization buffer for

single copy gene detection, (i.e. high sensitivity application) or

0.125 - 0.5 x 106 incorporated counts per ml of hybridization

buffer for high target work, for example colonies/plaques, PCR

products etc. Probe purification, to remove unincorporated

radioactive nucleotides, is strongly recommended.

Pre-wetting in a suitable buffer is essential for large blots

(>100cm2) or multiple blots. See Critical Parameters.

Hybridization may be carried out in bags, or boxes, provided

there is sufficient buffer for the container. Adequate circulation

of the buffer is essential. When hybridising several blots

together, the blot should move freely within the buffer.

Avoid placing the probe directly on the blots, as this will cause

excessive background.

Hybridization temperatures may vary with the probe. Lower

temperatures achieve lower stringency. The temperature of

hybridization used will depend on the degree of homology

between the probe and the target. 65-68°C is suitable for most

long probes (>100bases). With short/oligo probes (<50 bases)

hybridization temperature is usually defined as Tm-5°C: Tm

(melting temperature) = (4x number of G+C bases) + (2x

number of A+T bases)

Hybridization time can also vary. Short hybridization times

may be suitable for high target applications.

Stringency washes will depend on the nature of the probe and

target to be hybridized. Salt concentration and temperature

should be taken into consideration. The lower the salt

concentration, the greater the stringency. The higher the

washing temperature, the greater the stringency. Most

commonly, stringency washes proceed from "high salt"/"low

temperature", for example 5x SSC, 0.1% SDS at room

temperature, to "low salt/high temperature", for example 0.lx

SSC, 0. 1 % at 65°C (nominal hybridization temperature).

Some procedures include room temperature washes under

low stringency conditions. Do not allow the SDS to come out

of solution during these washes, significant levels of

background may result. Adequate circulation of the stringency

buffer is essential when washing. Washing in boxes is

advised.

10)

The use of SaranWrap with 35S labelled probes will

significantly increase exposure times. In this case the blot

should be air dried before autoradiography, if reprobing is not

required.

Hybridization in tubes

There are numerous commercially available rotisserie devices

suitable for use as hybridization ovens. These can accommodate 2

to 24 hybridization tubes. The major advantage of this approach to

hybridization is the use of low volumes of hybridization buffer, and

therefore minimal probe volumes. This is achieved because fluid is

able to move continually over the membrane.

Protocol

Prepare the hybridization buffer, for example

Denhardt’s Buffer

Modified Church Buffer

5x SSC

0.5M phosphate buffer, pH 7.2

5x Denhardt's solution

7% (w/v) SDS

0.5% (w/v) SDS

l 0mM EDTA

Ensure the SDS is in solution before use. Gentle

heating may be necessary

Combine all the components, make up to the required volume.

Prepare the radiolabelled probe using the appropriate

procedure.

Preheat the required volume of hybridization buffer to an

appropriate temperature.

Pre-wet the blot in a suitable dish, first in water then in an

appropriate buffer. Ensure that the nucleic acid side is

uppermost. Roll the blot along its length in such a way as to

minimise overlap in the tube. Place inside the hybridization

tube.

Add a small volume of appropriate buffer to the hybridization

tube, cap the tube. Unroll the blot by rotating the tube in the

opposite direction to the "rolled" blot.

Drain the tube of excess liquid and replace with the

appropriate volume of hybridization buffer.

Prehybridize for 30 minutes at the appropriate temperature.

Ensure that the tube is placed in the correct orientation within

the oven to avoid "rolling' up of the blot.

When using labelled double stranded probes, pipette the

required amount into a clean microcentrifuge tube. If the

volume is less than 2ml, make up to this volume with water or

TE buffer. Denature the probe by boiling for 5 minutes and

snap cool on ice. Briefly centrifuge to draw the contents to the

bottom of the tube

Add the probe to the pre-hybridization buffer.

10)

Hybridize overnight at the required hybridization temperature.

11)

Prepare the stringency wash solutions. The wash solution

should be used in excess. Use a volume that occupies

33-50% of the tube.

Low stringency wash;

2x SSC, 0.1% (w/v) SDS

Medium stringency wash:

lx SSC, 0.1% (w/v) SDS

High stringency wash:

0.lx SSC, 0.1% (w/v) SDS

12)

After the hybridization wash the blot as follows:

rinse briefly in 2x SSC, 0. 1 % (w/v) SDS

twice, 5 minutes each in 2x SSC, 0. 1 % (w/v) SDS

twice, 10 minutes each in lx SSC, 0.1% (w/v) SDS

four times, 5 minutes each in 0.lx SSC, 0.1% (w/v) SDS

13)

Remove the blot from the last stringency wash, drain and wrap

in SaranWrap and expose to X-ray film, for example Hyperfilm

MP. Keep the blot moist if it is to be reprobed.

Notes

There are a wide variety of hybridization buffers used by

researchers. This Denhardt's based buffer is that used in the

quality control of all Hybond nylon membranes. A reduced

concentration of SDS has been found to elevated

backgrounds following hybridization.. The Denhardt’s

hybridization buffer may be stored at -20°C if required.

This modification of the Church and Gilbert buffer is routinely

used at Amersham Pharmacia Biotech. It has been shown to

be suitable for Southerns, Northerns, dot blots and library

screening applications. The hybridization buffer may be

stored at room temperature. Ensure the SDS is fully dissolved

before use. This may be achieved with gentle heating.

Hybond-XL has been designed for use with very low volumes

of hybridization buffer (30-70µl/cm2). High backgrounds will

result if sub optimum volumes are used for the membrane and

hybridization conditions.

If there is significant overlap of the blot use of a nylon mesh

should be considered. The mesh achieves separation of the

blot layers allowing better probe access to these areas. It is

strongly advised that hybridization volume should be

increased (70-125µl/cm2). The nylon mesh should be at least

0.5cm larger than the blot. Place the mesh in the pre-wetting

solution before the blot, in subsequent manipulations treat as

"one". The nylon mesh may be reused after washing in 10%

(w/v) SDS and extensive rinsing in distilled water.

It is important not to allow air to become trapped between the

inner surface of the tube and the membrane. This can cause

areas of no signal or background following hybridization.

Hybond-XL has been designed for use with low volumes of

hybridization buffer (30-70µl/cm2). High backgrounds will

result if sub optimum volumes are used for the membrane and

hybridization conditions.

For radioactive applications use a probe concentration of 0.5 -

2 x 106 incorporated counts per ml of hybridization buffer for

single copy gene detection, i.e. high sensitivity application) or

0.125 - 0.5 x 106, incorporated counts per ml of hybridization

buffer for high target work, for example colonies/plaques,

PCR products etc. Probe purification, to remove

unincorporated radioactive nucleotides, is strongly

recommended.

Avoid placing the probe directly on the blot. Probe may be

added to the hybridization while the tube is in a vertical

position. If necessary probe may be mixed with a portion of

the hybridization buffer and added to the tube in a larger

volume.

10)

Hybridization temperatures may vary with the probe. Lower

temperatures achieve lower stringency. The temperature of

hybridization used will depend on the degree of homology

between the probe and the target. 65-68°C is suitable for

most long probes (>100bases). With short/oligo probes (<50

bases) hybridization temperature is usually defined as

Tm-5°C:

Tm (melting temperature) = (4x number of G+C bases) + (2x

number of A+T bases)

Hybridization time can also vary. Short hybridization times

may be suitable for high target applications

11)

Washing in boxes is much more effective and is

recommended if feasible. The inefficiencies of washing in

tubes may be overcome by increasing the number of

stringency washes while maintaining the same total wash

time.

12)

The use of SaranWrap with 35S labelled probes will

significantly increase exposure times. In this case the blot

should be air dried before autoradiography, if reprobing is not

required.