Adapted from Current Protocols in Molecular Biology
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.
- 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.
- 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.
- Wet the membrane carrying the immobilized RNA in 6X SSC.
- 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.
- 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.
- 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.
- 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.
- 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.
- Incubate at room temperature for 5 minutes. Pour wash solution into radioactive waste, replace wash solution and repeat.
- 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.
- 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.
- 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.
- 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.
- Perform autoradiography making sure to mark the orientation of the membrane on the film.
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.