Northern Blotting Protocol, Principle, Application, Result

Northern Blotting principle

In Northern Blotting the total RNA or mRNA is isolated from an organism of interest, and then electrophoresed on denaturing agarose gel, which separates the fragments on the basis of size. The next step is to transfer fragments from the gel onto nitrocellulose filter or nylon membrane.This can be performed by the simple capillary method.

The transfer or a subsequent treatment results in immobilization of the RNA fragments, so the membrane carries a semi permanent reproduction of the banding pattern of the gel. The RNA is bound irreversibly to the membrane by baking at high temperature (80°C) or by UV crosslinking. For the detection of a specific RNA sequence, a hybridization probe is used. A hybridization probe is a short (100-500bp), single stranded nucleic acid either DNA or RNA probe that will bind to a complementary piece of RNA.

Hybridization probes are labeled with a marker (radioactive or non-radioactive) so that they can be detected after hybridization. In non-radioactive detection the probe is labeled with biotin or digoxigenin. The membrane is washed to remove a non-specifically bound probe and the hybridized probe is detected by treating the membrane with a conjugated enzyme, followed by incubation with the chromogenic substrate solution. As a result a visible band can be seen on the membrane where the probe is bound to the RNA sample.

Northern Blotting Steps

The Northern Blotting is divided into following steps:

Denaturing Agarose Gel Electrophoresis

• This method is used to separate RNA molecules according to their molecular size.
• In this method, formaldehyde (a denaturant) is used along with MOPS electrophoresis buffer. 
• RNA has a high degree of secondary structures, making it necessary to use denaturing gels.
• Formaldehyde in the gel disrupts the secondary RNA structures so that RNA molecules can be separated by their charge migration. 
• For analysis of RNA molecules, 1 to 1.2% agarose gels are used depending on the size of RNA to be separated. 
• The position of RNA in the agarose gel is visualized by staining with low concentration of fluorescent intercalating dyes, such as Ethidium bromide. This can be added either in the gels or in the RNA sample before loading for better resolution. 
• The integrity and size distribution of total RNA can be checked by observing the stained RNA.

Northern blotting technique

• Northern blotting is the capillary transfer of resolved RNA fragments from the denaturing agarose gel to the nitrocellulose/nylon membrane. 
• In this upward capillary transfer procedure, a support is used which is placed in a reservoir of transfer buffer to elevate the entire assembly. 
• The wet wicks are placed on the support with both ends completely dipped in transfer buffer.
• A gel is placed on the wicks with RNA transferred side facing down.
• Over the gel, a wet positively charged nylon membrane is placed. 
• A stack of paper towel is kept on filter papers. 
• A small weight is placed over this entire assembly and is kept overnight.
• During the capillary transfer the RNA bands are transferred to positively charged nylon membranes in presence of a specific buffer. 
• The resolved RNA fragments are transferred to the corresponding positions on the nylon membrane after the capillary transfer. 
• The RNA is then immobilized on the membrane either by baking at high temperature or UV crosslinking. This results in the covalent linkage of RNA to the membrane, which prevents the nucleic acid from being washed away during the subsequent processing. This is followed by hybridization with labelled DNA or RNA probe and then the RNA of interest is detected on the membrane. 

Detection

• After capillary transfer, RNA bands bound to the membrane are detected using a chromogen. The RNA of interest is hybridized with a biotinylated probe specific to it. 
• The membrane is washed to remove the excess unbound probes. It is then treated with Horseradish peroxidase (HRP)-conjugated streptavidin which attaches to the hybridized RNA. Finally, the membrane is incubated in a substrate solution containing TMB/ H2O2 (Tetramethyl benzidine H2O2 substrate) that reacts with HRP and as a result a corresponding blue-colored RNA band develops on the nylon membrane.

Material Required

• Total RNA
• Hybridization Buffer
• Biotinylated Probe
• RNA Loading Buffer
• Wash Buffer I
• Wash Buffer II
• Blocking Powder
• TMB/H2O2
• Streptavidin HRP Conjugate
• Conjugate Dilution Buffer
• MOPS Electrophoresis Buffer
• Transfer Buffer
• Tween 20
• Agarose
• Formaldehyde
• Filter Paper
• Wicks
• Nylon Membrane
• Blotting Sheet
• Sterile Disposable Petri plates
• Glassware: Conical flasks, Beakers
• Reagents: Distilled water, Ethidium bromide (10 mg/ml), Isopropanol, Ethanol
• Other: Glass plate, Plastic box, Plastic tray, Gel rocker, Micropipettes, Tips, Microwave/Burner/Hotplate, Hot Air Oven, Incubator Shaker (55o C and 65 oC), Forceps, Crushed ice.

Preparation of Hybridization Buffer: Add 0.1 g of blocking powder to 10 ml of hybridization buffer. Mix well before use.

Preparation of Streptavidin HRP-Conjugate Buffer: Add 9 μl of Tween 20 in 9 ml of conjugate dilution buffer. Add 3 μl of Streptavidin-HRP Conjugate in 9 ml of conjugate dilution buffer for each experiment just prior to use.

Northern Blotting Protocol

Day 1: Denaturing Agarose Gel Electrophoresis

1. Preparation of 1X MOPS Electrophoresis Buffer: To prepare 500 ml of 1X MOPS Electrophoresis Buffer, add 50 ml of 10X MOPS Buffer to 440 ml of RNase free water and add 10 ml of Formaldehyde (37%). Mix well before use.
2. Preparation of Denaturing Agarose gel: To prepare 50 ml of 1.2 % agarose solution, mix 5 ml of 10X MOPS Electrophoresis Buffer with 45 ml of autoclaved deionized water in a glass beaker or flask. To this add 0.6 g of agarose. Heat the mixture in a microwave, burner or hot plate, swirling the glass beaker/ flask occasionally, until agarose dissolves completely (Ensure that the lid of the flask is loose to avoid buildup of pressure). Allow solution to cool to about 55- 60o C. Add 0.9 ml of 37% Formaldehyde and mix well and add 1μl of EtBr (10 mg/ml), mix well and pour the gel solution into the gel tray sealed on both sides with adhesive tape. Allow the gel to solidify for about 30 minutes at room temperature (15-25o C). 

NOTE: Before running the gel, equilibrate it in 1X MOPS Buffer for atleast 30 minutes.

3. Loading of the RNA samples: To prepare sample for electrophoresis, add 2 μl of 5X RNA gel loading buffer to 10 μl of total RNA samples. Mix well by pipetting and load the samples into 5 wells. 
4. Electrophoresis: Connect the power cord to the electrophoretic power supply according to the conventions: Red-Anode and Black- Cathode. Electrophorese at 100 volts and 70 mA until dye markers have migrated an appropriate distance, depending on the size of RNA to be visualized.

NOTE: Toxic fumes are released on electrophoresis, hence it is necessary to run the gel in a fume hood.

Capillary Transfer of RNA

1. After electrophoresis, soak the gel in RNase-free water for 5 minutes to remove formaldehyde.
2. Repeat the above step twice and then discard water.
3. Observe the gel under UV transilluminator to excise the gel piece using a gel cutter.

NOTE: Excise the entire piece of gel i.e. 5 lanes. Take care not to excise the individual lanes. Do not expose yourself to UV.

4. Set up capillary blot with transfer buffer as follows.
   • Rinse the materials required for transfer with isopropanol followed with RNase-free water thoroughly.
   • Fill the buffer reservoir with 200 ml 10X transfer buffer.
   • Keep support in the buffer reservoir.
   • Wet the wicks with a transfer buffer. Ensure both the ends of the wicks are completely dipped in the transfer buffer.
   • Place the gel piece upside down i.e. RNA transferred side should face the wick.
   • Wet the nylon membrane for few minutes in the transfer buffer. Place the membrane supplied on the gel. Ensure that no air bubbles are trapped between the gel and the membrane, as this will affect efficient transfer.
   • Wet the filter paper and place over the membrane, ensure no air bubbles are trapped between the membrane and filter paper.
   • Cut blotting sheets of the same size and stack over filter paper.
   • Carefully place a weight over this stack. Ensure that the book is placed on the centre of this stack, such that even pressure is applied.
   • Carry out transfer overnight at room temperature.

Day 2: Immobilization of RNA on membrane

1. Carefully remove the stack of blotting paper and filter paper after overnight transfer.

NOTE: Mark the side of the membrane that faced the gel as this is the right side i.e. RNA transferred side.

2. Observe the membrane and the gel piece under UV transilluminator to check whether the complete transfer has taken place.
3. Expose the membrane to UV light for 5 minutes (placed between the inner layers of tissue paper). This helps in fixing the RNA to the membrane.
4. Switch off the UV, turn over the membrane and expose it to UV light for another 5 minutes.
5. Bake the membrane at 70-80 oC for 30 minutes (place the membrane between the inner layers of tissue paper)
6. Place the membrane in a ziplock or autoclaved petri plate at 4 oC.
7. Mark the lanes on the membrane with a pencil, cut along the length of the membrane.
8. Use one strip for hybridization and development and the remaining strips can be stored at 4 oC until further use.

Hybridization

1. Set an incubator shaker at 55 oC, prior to placing the membrane for Prehybridization.
2. Place the membrane (RNA transferred side facing down) in a petri plate containing 10 ml of hybridization buffer.
3. Carry out Prehybridization at 55 oC for 1 hour, with constant shaking (70-80 rpm).
4. Keep 1 vial of biotinylated probe for 5 minutes in boiling water bath and immediately chill by placing it on ice for 5-10 minutes.
5. Remove petri plate and discard the buffer.
6. Add 10 μl of this probe to the 10 ml of Hybridization buffer in the petri plate. Mix thoroughly and add drop wise to the petri plate. Make sure that you don’t add probe directly on the membrane.
7. Seal the petri plate and incubate at 55 oC in an incubator shaker overnight with mild shaking at about 70-80 rpm.

Day 3: Washes and Detection

1. Transfer the membrane into a fresh petri plate containing 10 ml of Wash Buffer I.
2. Gently swirl the petri plate for 15 minutes at room temperature. Repeat the wash one more time. Discard the buffer after each wash.
3. Add 10 ml of prewarmed Wash Buffer II (65 oC) and gently swirl the petri plate. Incubate at 65o C for 15 minutes in an incubator shaker and gently swirl. Repeat this step. Discard the buffer after each wash.

NOTE: Do not let the membrane go dry at any step.

4. Add 9 ml of Streptavidin-HRP conjugate buffer (Refer Important instructions) to the petri plate and incubate at room temperature for 30 minutes with gentle rocking. Discard the conjugate buffer.
5. Take 30 ml of conjugate dilution buffer in autoclaved test tube and add 30 μl of Tween 20 to it and mix it thoroughly. This is to be used for further washes.
6. Carry out washes in a fresh and dried petri plate. Do not carry out washes in the petri plate used for conjugation. Use 10 ml of conjugate dilution buffer (See step 5) to carry out washes of 5 minutes at room temperature. Repeat the above step two more times.
7. Add 5 ml of TMB/H2O2 and gently swirl at room temperature until a blue colour band develops.
8. After blue colour band is seen stop the reaction by placing the membrane in distilled water.

Observation and Result

• Lane 1: RNA sample on 1.2 % denaturing agarose gel
• Lane 2: After Northern hybridization a blue band develops on the nylon membrane 

Precautions

• Prior to start the experiment, the electrophoresis tank should be cleaned with detergent solution (e.g., 0.5% SDS), thoroughly rinsed with RNase-free water, and then rinsed with ethanol and allowed to dry.
• Tips, pipettes, electrophoresis unit etc to be used for the experiment must be UV treated for 15-20 minutes.
• Use sterile, disposable plasticwares and micropipettes reserved for RNA work to prevent cross-contamination with RNases from shared equipments.
• Use RNase-free water for diluting the solutions.

Application of northern blotting

• Used for Analysis of gene expression.
• Used to show overexpression of oncogenes and downregulation of tumor-suppressor genes in cancerous cells.
• Used to analyze the gene expression in the rejection of transplanted organs.

Northern Blotting Advantage

• It can detect small changes in gene expression.
• It has a high specificity.
• Can detect the RNA size.
• The observation of alternate splice products. 
• The use of probes with partial homology.
• The quality and quantity of RNA can be measured on the gel prior to blotting.
• The membranes can be stored and reprobed for years after blotting.

Northern Blotting Disadvantage

• Used to study one or a small number of genes. It can not visualize thousands of genes at a time.
• In Northern Blot the sample is often degraded by RNases.
• The chemicals used in most northern blots can be a risk to the researcher such as formaldehyde, radioactive material, ethidium bromide, DEPC, and UV light.
• Has a low sensitivity as compared to RT-PCR.

References