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:

.	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.

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.