Northern blotting - gel preparation and treatment(Northern杂交-凝胶制备及处理)

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

1)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.

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

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.

3)Separate the RNA samples using lx MOPS buffer as the electrophoresis buffer.

4)Following electrophoresis, if appropriate, visualize the RNA within the gel with UV light and photograph.

5)Place the gel in a suitable tray or dish and cover with distilled water. Incubate the gel with gentle agitation for 15 minutes.

6)Discard the water and replace with sterile 10x SSC. Agitate for 15 minutes. Repeat this step once more.

7) Set up the capillary blot as described.

Notes

1)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.

3)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.

4) 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.

5) 10x SSC or 20x SCC can be used as the transfer buffer.

Glyoxal protocol

1) 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.

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

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.

3)Separate the RNA samples using lx MOPS buffer as the electrophoresis buffer.

4) Following electrophoresis, if appropriate, visualise the RNA within the gel with UV light and photograph.

5) Set up the capillary blot as described using a neutral transfer buffer.

Notes

1) 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.

2) Glyoxylated RNA should be run more slowly than formaldehyde gel to prevent formation of pH gradients.

3) Glyoxal gels may be electrophoresed in 1x MOPS thereby eliminating the need to recirculate the buffer.

4) Glyoxal may interact with ethidium bromide altering the dyes spectral properties.

5) 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

1)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.

2)Prepare the radiolabelled probe using the appropriate procedure

3)Preheat the required volume of hybridization buffer to the appropriate temperature.

4)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).

5)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

6)Add the probe to the pre-hybridization buffer.

7)Hybridize overnight with gentle agitation at the required temperature.

8)Prepare the stringency wash solutions. The wash solution should be used in excess, at least 1-5 ml/cm2 of membrane.

High stringency wash:
0.lx SSC, 0.1% (w/v) SDS

9) 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

1)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.

2) 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.

3) 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.

4) Avoid placing the probe directly on the blots, as this will cause excessive background.

5) 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.

6) 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).

7) 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.

8) 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

1)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.

2) Prepare the radiolabelled probe using the appropriate procedure.

3) Preheat the required volume of hybridization buffer to an appropriate temperature.

4)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.

5)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.

6)Drain the tube of excess liquid and replace with the appropriate volume of hybridization buffer.

7) 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.

8) 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

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:
a)rinse briefly in 2x SSC, 0. 1 % (w/v) SDS
b)twice, 5 minutes each in 2x SSC, 0. 1 % (w/v) SDS
c)twice, 10 minutes each in lx SSC, 0.1% (w/v) SDS
d)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

1) 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.

2) 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.

3) 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.

4) 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.

5) 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.

6) Hybond-XL has been designed for use with low volumes of hybridization buffer (30-70µl/cm2).

7) High backgrounds will result if sub optimum volumes are used for the membrane and hybridization conditions.

8) 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.

9) 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 asTm-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.