Northern Blot Hybridization

Adapted from Current Protocols in Molecular Biology

Objective:

The hybridization step of a northern blot allows identification of RNA blotted and immobilized on a membrane with a radioactive (or otherwise labeled) nucleic acid probe.

Required Materials

  • Membrane (nylon or nitrocellulose) with immobilized RNA.
  • Labeled DNA or RNA probe.
  • 20 mM Tris-HCl (pH 8.0).For blots from glyoxal gels.
  • 20X SSC
  • 20% SDS
  • formamide prehybridization/hybridization solution.

Procedures

  1. If the membrane was blotted from a glyoxal gel, soak the membrane in 20 mM Tris-HCl (pH 8.0) for 5 minutes to disassociate the glyoxal from the bound RNA.
  2. Wet the membrane carrying the immobilized RNA in 6X SSC.
  3. Place the membrane in a hybridization tube with the RNA-side facing in, and add ~1mL formamide prehybridization/hybridization solution per 10 cm2. Alternatively one can use resealable bags. In which case, the membrane should be sealed into a bag after being wetted. Then a corner of the bag can be cut off to allow pipetting of the prehybridization/hybridization solution into the bag. The small corner can then be resealed.
  4. Place the tube in a hybridization oven and incubate with rotation 15 minutes (3 hours if using a nitrocellulose membrane) at 42°C (DNA-probe) or 60°C (RNA-probe). If using resealable bags, it can be shaken or rocked slowly in a suitable incubator or water bath.
  5. If the probe is dsDNA, denature by boiling for 5 minutes, and immediately place it on ice. Denatured probe should be added to the hybridization solution as soon as possible after denaturation.
  6. Pipet the desired volume* of probe into the hybridization tube and continue to incubate with rotation overnight at 42°C (DNA-probe) or 60°C (RNA-probe).   If using resealable bags the probe can be added by inserting a syringe into an uncut corner and injection of the probe. The bag must then be carefully be resealed to avoid leaking radioactivity.
    * The probe concentration in the hybridization solution should be 10 ng/mL if the specific activity is 108 dpm/µg or 2 ng/mL if the specific activity is 109 dpm/µg.
  7. Pour off the hybridization solution into the radioactive waste, and replace with at least equal volume of 2X SSC/0.1% SDS. If using resealable bags, membrane can be transferred to a plastic dish for the following washes.
  8. Incubate at room temperature for 5 minutes. Pour wash solution into radioactive waste, replace wash solution and repeat.
  9. Low stringency wash: Replace wash solution with 0.2X SSC/0.1% SDS and incubate for 5 minutes with rotation at room temperature. Change wash solution and repeat.
  10. Medium stringency wash (optional): Change wash solution with prewarmed (42°C) 0.2X SSC/0.1% SDS and incubate with rotation at 42°C for 15 minutes. Repeat once.
  11. High stringency wash (optional): Change wash solution with prewarmed (68°C) 0.2X SSC/0.1% SDS and incubate with rotation at 68°C for 15 minutes. Repeat once.
  12. Remove final wash and rinse membrane in 2X SSC at room temperature. Blot excess liquid and cover in UV-transparent (SaranWrap) plastic wrap. Do not allow membrane to dry out if it is to be stripped and reprobed.
  13. Perform autoradiography making sure to mark the orientation of the membrane on the film.

Recipes

Formamide Prehybridization/Hybridization Solution

  • 50% (v/v) Formamide
  • 5X SSC
  • 5X Denhardt's Solution
  • 1% (w/v) SDS
  • Add 100µg/mL denatured salmon sperm DNA (or Herring testes DNA) just before use.