Northern hybridization
Summary
RNA samples transferred and immobilized to membranes can be hybridized to specific probes that can be used to localize the RNA of interest. Depending on the experimenter and conditions, any one of a variety of methods can be selected to label and detect the probe. After treating the membrane with a blocking reagent that inhibits nonspecific uptake of the probe, incubating the membrane with the probe under conditions suitable for hybridization of the probe to the immobilized target RNA, and then washing the membrane extensively to remove the nonspecifically bound probe, ultimately produces a distribution of images of tightly bound probes on the membrane. After analyzing the hybridization results, the probes can be washed off the membrane and the membrane can be reused for the next experiment. This experiment is based on the "Guide to Molecular Cloning, Third Edition", translated by Peitang Huang et al.
Operation method
Northern hybridization
Principle
RNA samples transferred and immobilized to membranes can be hybridized to specific probes that can be used to localize the RNA of interest. Depending on the experimenter and conditions, any one of a variety of methods can be selected to label and detect the probe. After treating the membrane with a blocking reagent that inhibits nonspecific uptake of the probe, incubating the membrane with the probe under conditions suitable for hybridization of the probe to the immobilized target RNA, and then washing the membrane extensively to remove the nonspecifically bound probe, ultimately produces a distribution of images of tightly bound probes on the membrane. After analyzing the hybridization results, the probes can be washed off the membrane and the membrane can be reused for the next experiment.
Materials and Instruments
DNA or RNA probes
Pre-hybridization solution SSC
Filter paper Boiling water bath Water bath
Move
I. Materials
1. Buffers and solutions
Pre-hybridization solution (0.5 mol/L pH 7.2), 7% (m/V) SDS, 1 mmol/L EDTA ( pH 7.0))
SSC ( 0.5X, 1X, and 2X) containing 0.1% (m/V) SDS
SSC (0.1X and 0.5X) with 0.1% (m/V) SDS
2. Nucleic acids and oligonucleotides
DNA or RNA probes (> 2X108 cpm/μg)
RNA immobilized on membranes
3. Specialized equipment
Filter paper (Whamian 3 MM or equivalent)
Boiling water bath
Water bath preheated to 68°C
Water bath preset to hybridization temperature
II. Methods
1. Incubate the membrane at 68℃ for 2 h in 10~20 ml of prehybridization solution.
2. If using a double-stranded probe, heat 32P-labeled double-stranded DNA at 100℃ for 5 min to denature, then quickly transfer to ice water for cooling.
3. Add the denatured or single-stranded radiolabeled probe directly into the prehybridization solution and continue to incubate at the appropriate temperature for 12 to 16 h.
4. After hybridization, remove the membrane from the plastic bag and quickly transfer it to a plastic cassette containing 100~200 ml of 1X SSC, 0.1% SDS at room temperature, cover the cassette and place it on a horizontal oscillator for 10 min of gentle shaking.
5. Transfer the membrane to another plastic box containing 100~200 ml of 0.5X SSC containing 0.1% SDS preheated to 68°C. Shake gently at 68°C for 10 min.
6. Repeat step 5 for at least 2 more washes, making a total of 3 washes at 68°C.
7. Dry the membrane on blotting paper and allow the membrane to radiographically self-develop on X-rays (Kodak XAR-5 or equivalent) in a dark box with a sensitizing screen at -70°C for 24-48 h. Alternatively, the image on the membrane can be scanned with a phosphor imaging system. 
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