Northern Blot protocol

Reagents

All buffers need to be prepared with DEPC-treated (and autoclaved) distilled water and kept in Rnase-free glassware.

10x BPTE buffer (Gel and electrophoresis buffer)

100 mM PIPES

300 mM Bis-tris

10 mM EDTA, pH 8

Add 15 g PIPES, 30 g Bis-Tris and 10 ml 0.5 M EDTA to 450 ml of DEPC-treated water. Adjust volume to 500 ml. Dissolve 10X buffer to 1X before use.

NorthernMax Glyoxal gel loading solution (RNA sample preparation)

Purchase from Ambion (catalog # 8551). Use with caution as it contains DMSO, ethidium bromide and glyoxal.

20X SSC (transfer buffer, washes, etc…)

Dissolve 175.3 g NaCl and 88.2 g Sodium Citrate in 800 ml of Rnase-free water. Adjust to pH 7 (adding HCl). Bring volume to 1 l and autoclave.

20% SDS (membrane washes, gel box treatment, etc…)

Dissolve 100 g of SDS in 500 ml of Rnase-free water.

Modified Church buffer (pre-hybridization and hybridization)

0.36 M Na2HPO4

0.14 M NaH2PO4

1 mM EDTA

7% SDS

For 500 ml of buffer, dissolve first 25.56 g Na2HPO4 in 200 ml of Rnase-free water. Add 1 ml of 0.5 M EDTA. Add 8.4 g of NaH2PO4. If difficult to dissolve, heat the solution. Add 175 ml 20% SDS; heat the solution until all the components are dissolved, this may take time. Bring final volume to 500 ml and store at 37° C to avoid precipitation of salts at room temperature.

Procedure

Gel electrophoresis

Make sure gel box, tray and combs are Rnase-free. Several methods can be used. Soaking all the components in 1% SDS overnight then rinsing with Rnase-free water works well.

  1. Mix appropriate volume of total RNA (at least 10 μg) with an equal volume of Northernmax glyoxal dye. If RNA is too dilute, as little as 0.5 volumes of dye can be used.
  2. Heat samples at 50° C for 30 minutes, then chill on ice for a few minutes. If less than 1 volume of dye was used, extend incubation to 1 h.
  3. While samples are incubating, prepare 1.4% agarose gel with 1X BPTE buffer.
  4. Load samples into wells and run gel at 80 V until the bromophenol blue dye marker reaches 2/3 of the length of the gel.
  5. Take picture of gel under UV light.

RNA transfer to nylon membrane

  1. If necessary, trim gel removing unused lanes. Measure gel dimensions with a ruler. Soak gel in Rnase-free water.
  2. Cut piece of membrane the size of the gel. Soak membrane in 20X SSC buffer.
  3. Assemble capillary blot unit: get a pyrex dish (Rnase-free) and fill with 300 ml of 20X SSC. Place a horizontal support on pyrex dish. Lay a sheet of 3 MM Whatman paper across the support, with ends of filter paper soaking in 20X SSC. Make sure the whole filter paper is soaked on 20X SSC solution. Cut a stack of paper towels to the size of membrane.
  4. Cut a piece of agarose in the upper left corner of the gel to orient the position of the first sample. Invert gel and place it on top of the blotting support.
  5. Carefully lay the nylon membrane on top of the gel; remove bubbles by pressing with a 10 ml plastic pipette. Clip off the side corner of the membrane as did with the gel.
  6. Place strips of saran wrap around the gel to avoid buffer flow around the gel.
  7. Place two sheets of 3MM whatman paper, cut to the size and soaked in 20 X SSC, on top of membrane.
  8. Place the stack of paper towels of about 10 cm high on top of gel and membrane. Place a glass plate on top of the towels and top with a moderate weight.
  9. Transfer for about 9 h or overnight.
  10. After blotting, remove the membrane carefully, place it RNA side up on a filter paper and dry completely at room temperature for about 2 h.
  11. Wrap the dried membrane in saran wrap and expose it, RNA side down, to a UV transilluminator for 60 s, approximately, to cross-link RNA to membrane.

Pre-hybridization and hybridization

Use of radioactive material must be done after completion of the OSU radiation safety online course AND after proper training in the lab by a certified individual. Details of the procedures for handling radioactive materials will not be included in this protocol. It is assumed that every user of this protocol has received the correct training for 32P use and for disposal of radioactive waste.

  1. HeatChurch buffer at 65° C in a water bath.
  2. Wet membrane in Rnase-free water and put it in the hybridization bottle. Add 10-20 ml of Church buffer and place bottle in the oven, with rotation, at 65° C for pre-hybridization. Pre-hybridize for at least 2 hours.
  3. Radiolabel the probe using your preferred method. Usually, use 20-25 ng of DNA or cDNA and 3μl of 32P-tagged dCTP for labeling. See labeling protocol at end of this page.
  4. After labeling is done, denature the labeled DNA in a boiling water bath for 5 min. Chill on ice for a few minutes.
  5. Dump pre-hybridization solution and add fresh Church buffer to the bottle containing the membrane. Add the denatured probe to the buffer and hybridize overnight in oven at 65° C.

Stringency washes

  1. Dump hybridization buffer in the liquid radioactive waste container.
  2. Wash membrane for 5 min at 65° C with 1X SSC/0.5% SDS. Dump wash solution in the liquid radioactive waste container and repeat wash with 1X SSC/0.5% SDS.
  3. Wash membrane for 15 min at 65° C with 0.5X SSC/0.5% SDS. Dump wash solution in hot sink and repeat wash with 0.5X SSC/0.5% SDS.
  4. After final rinse, remove membrane from bottle and air dry for a few minutes.
  5. Wrap membrane in saran wrap and expose it to a phosphor screen in cassette, RNA side up. Leave cassette at room temperature for 2-6 hours or overnight. Follow protocol for scanning the screen in the phosphorimager.

Radioactive labeling of DNA probe with Redi-Prime kit (Amersham)

  1. Mix DNA (20-25 ng) and water to a final volume of 47 μl. Denature DNA in boiling water bath for 5 min, then chill on ice.
  2. Add denatured DNA to Redi-prime labeling solution and mix well.
  3. Add 3 μl of dCTP- 32P radionuclide to the tube containing labeling mix with probe. Incubate at room temperature for 1-2 h.
  4. Denature labeled probe in boiling water bath for 5 min; chill on ice. Add denatured, labeled probe to hybridization solution.

Prepared by Miguel Vega-Sanchez

343 Kottman Hall, Plant Path OSU