PROTOCOL FOR NORTHERN BLOTTING

Developed by: Christopher Todd Hittinger Modified from: "Bio-Rad Vacuum Blotter Instruction Manual" and "Analysis of RNA by Northern and Slot Blotting Hybridization" from Short Protocols in Molecular Biology Preparation 1. EVERYTHING must be Thoroughly cleaned of RNAses, and uLoves must be worn at all times. 2. Use RNAse-ZAP or some other commercially available RNAse cleaner to clean the work area and all equipment that may come into contact, directly or indirectly, with the RNA. If none are available, use a dilute solution of bleach. 3. Clean everything with 95 % ethanol. This will also help any residual RNAse-ZAP evaporate. 4. Put sign(s) around the work area that stress(es) the importance of a lack of contamination. 5. Also, use only DEPC-treated (diethylpyrocarbonate) chemicals. 6. Chemicals can be DEPC-treated by adding 2 mL of DEPC per L of solution, followed by autoclaving. Pouring the Formaldehyde Gel (for a 1 % gel) 7. Put 0.50 g agarose into an Erlenmeyer flask. 8. Add 36 mL of DEPC-H20. 9. Add-5 mL of DEPC-10x MOPS. 10. Add 9 mL of 12.3 M (37 %) formaldehyde (does not need DEPC-treatment). 11. Boil in microwave to dissolve agarose. BE CAREFUL to inhale as little of the formaldehyde fumes as possible. 12. Pour gel into set up box with comb in it. If using 66 p-L loading samples as described here, 3 wells must be taped together to form I using the 8-welled comb in order for the sample to fit. For any sized loading,- sample, DO NOT use the outer wells. Loading the Gel (66 uL sample described, but ratios are what is important) 13. Dilute 30 g of quantified tRNA to 12.1 uL in DEPC-H20. 14. Ado 5.5 uL of 10x MOPS 15. Add 9.9 uL of 12.3 M formaldehyde. 16. Add 27.5 L of formamide. 17. Incubate in a PCR machine for 15 minutes at 55 C [program 16 (I x cycle 3 1) in PCR machine in Dr. Gathman's lab, assuming it has not been changed]. 18. Add 11 L of formaldehyde running buffer (RNA loading dye). 19. Add to appropriate well, avoiding outer wells. Running the Gel 20. Perform electrophoresis at 100 volts for 1 hour or until loading dye has run 2/3 to of the way through the gel. 21. 1 have NOT established whether or not staining interferes with the blotting, procedure, so do not do it unless you wish to test whether or not it does interfere with the procedure. 22. If you do stain, use ethidium bromide in DEPC-0.5 M ammonium acetate. 23. For destaining, use DEPC-0.5 M ammonium acetate. 24. Before moving gel, let gel cool a little, or it may break because formaldehyde, gels are much weaker than normal agarose gels. Even after cooling, be exceedingly careful. Vacuum Blotting 25. Rinse the gel in a dish with DEPC-H20. 26. While rinsing, set up the vacuum blotter (clean it for RNAses first) (27-37). 27. Attach a water trap (side-arm flask) to the vacuum blotter with a tube. 28. Attach the water trap to the provided regulator port (thing with the pressure gauge) with a tube. 29. Attach the regulator port to the vacuum (huge object on wheels [actually a freeze-drier]) with a tube.

Cut a piece of Whatman 3mm paper and a piece of Hybond N+ nylon membrane paper on one of the gel patterns (there are several in Dr. Lilly's lab on the first shelf over the main bench). Be especially careful to keep these RNAse-free. 31. Wet the membrane in DEPC-H20. 32. Wet the membrane and the filter paper in DEPC-10X SSC. 33. Put the porous vacuum plate on the main stage of the vacuum blotter. 34. Put the wetted filter paper on the porous vacuum plate in the center with long ends of each rectangle parallel to each other. 35. Put the wetted nylon membrane on top of the filter paper. 36. Wet the seal o-ring (on the main stage of the vacuum blotter) with DEPC-H20. 37. Put the window gasket that has been precut for the appropriate sized gel on next- The window gasket must overlap the nylon membrane by at least 5 mm, and it should be consistent on all sides. You will probably have to adjust the positioning of the filter paper and the nylon membrane. The window gasket must also cover the entire reservoir seal o-ring to ensure a vacuum (Figure 3-2 on page 6 of the Auto-Rad Vacuum-Blotter Instruction Manual" is helpful in illustrating the last several steps). 38. Place the gel on the nylon membrane such that the edges are overlapped by the window gasket. Be certain that all of the area containing RNA is touching the nylon membrane, but do not move the gel once it touches the nylon membrane. 39. Place the sealing frame over the entire setup. 40. Unscrew the bleeder valve on the vacuum regulator. 41. Hold the gel in place with your gloved hands, while another person turns on the vacuum. 42. Have them adjust the bleeder until the vacuum regulator reads a pressure of 5 inches Hg, while you hold the gel in place. 43. While holding the gel in place, add 1 L of DEPC-10X SSC to the transfer reservoir so that the gel is completely covered by the transfer solution. 44. Put the lid on. 45. Allow transfer to occur at 5 inches Hg, for 1.5-2.5 hours. Effective transfer has not been established outside this pressure or time of transfer range, but the instruction manual contains information suggesting deviation from this protocol is detrimental to effective transfer. Preparation for Probing 46. Disconnect the vacuum from the vacuum blotter, and turn the vacuum off 47. Remove the sealing frame and allow the transfer solution to drain off. 48. Remove the gel. 49. If you wish to estimate transfer efficiency and determine whether RNA was present in the gel to begin with (all of it will not have transferred), you may stain it with ethidium bromide inDEPC-0.5 M acetate and destain it in DEPC 0.5 ammonium acetate 30. 50. 51. 52. 53. Remove the window gasket, and remove the nylon membrane. Soak the membrane in DEPC-2x SSC for 5 minutes or just rinse in DEPC-2x SSC. Wrap the blot in Saran Wrap. UV-crosslink both sides of the blot in the UV-crosslinker in Dr. Champine's laboratory. After crosslinking, RNAse precautions are no longer necessary for the blot. 54. Rinse all of the blotting equipment with DEPC-H20 and dry it. Store all the equipment in an out-of-the-way place not likely to be disturbed by RNAse-laden people. Put these things in plastic if possible. Probing 55. For the most part, the Northern blot can be probed as normal. 56. In order to be certain of transfer, each northern blot should first be probed with a probe of the "gene" encoding 18s rRNA. These probes will work for at least I month. 57. Perform all washes as normal (2 1st washes at room temperature and I 2nd wash at 65 C). Put on film for about 1 hour for a fresh probe and proportionately less for older probes.

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After developing, trace the position of the northern blot onto the developed film, so that you will know where the ribosomal bands are for future probes. There should be 1 visible band. 59. The darkness of these bands can be used a loading control, 60. Stop the northern blot with boiling 0.5 % SDS, and allow it to cool to room temperature. 61. Put it on film for 3 days to ascertain the efficiency of stripping. 62. Probe with a probe of the desired gene as normal. 63. Only perform the 1st wash 2x at room temperature. 64. Put on film for 1 day to 1 week depending on circumstances. Generally, 3 days is optimal. 65. Develop and trace the position of the northern blot onto the film, and compare it with the film from when the northern blot was probed with the ribosomal RNA probe. Some nonspecific binding will probably occur at the location of the ribosomal band, so ignore that and look for other bands. There will also probably be a band toward the lower end of mRNA length. It is not yet known what this band corresponds to, but it has been present every time northern blots have been probed with cDNA probes. 66. The northern blot may be probed with other probes after stripping it, but after stripping it 4 times, there is a significant loss of efficiency of probe binding, probably because some of the RNA is removed with each time it is stripped.