Northern blotting technique

Electrophoretically separated RNA molecules get transferred from the gel to the absorbent sheet, immersed in a labeled probe for hybridization. Thus, the northern blotting follows the same steps of Southern blotting. However, RNA instead of DNA gets blotted. The standard Northern blotting procedures help to compare the quantities of the transcripts from different tissues. The technique’s sensitivity increases to a 100 fold using an mRNA or a messenger RNA. The technique analyses many types of RNAs such as micro RNA, small nuclear RNAs or snRNAs, small interfering RNAs or siRNAs, and mRNA. The technique is helpful in RNA interference studies or RNAi technology. It also involves studying the expression of oncogenes. Northern blotting includes human RNAs, plant, and animal RNAs. The technique has placed itself very well in genetic research and engineering.

Image: Northern blotting

The description of the blotting procedure is as follows:

Step 1: Isolation of mRNAs:

RNA extraction uses a homogenized tissue sample. Oligo (dT) cellulose chromatography helps in isolating mRNA with a poly (A) tail. We know the mechanism of polyadenylation well. It protects the RNA from degradation by adding polyadenine nucleotides to the RNA molecule. Widely used methods include phenol-chloroform extraction or trizol method.

Step 2: Electrophoresis:

Once the RNA isolation gets completed, the loading of the samples in the wells helps to obtain the bands. Separation of the RNA molecules involves agarose gels most of the times. Fragmented RNA or micro RNA separation may include polyacrylamide gels. Formaldehyde acts as a denaturing agent in the electrophoresis. It limits the RNA secondary structure. Fragment sizes comparison uses a ladder RNA in another well. Electrophoresis involves staining the gel with ethidium bromide. Handling ethidium bromide requires caution because of its carcinogenicity and neurotoxicity. Ethidium bromide intercalates the RNA, thereby acting as an intercalating agent.

Step 3: Using a nylon membrane:

After separation of RNA molecules through gel electrophoresis, the samples get transferred to a nylon membrane through a capillary system. Following is the apparatus for northern blotting:

The first step involves filling a tray with a buffer solution consisting of formamide. It helps in lowering the annealing temperature of the probe-RNA interaction thereby preventing RNA from getting degraded. Then the RNA gets immobilized to the membrane through a covalent linkage. UV light or heat facilitates this property. The blotting paper used in this apparatus plays a role in capillary action by carrying the buffer solution through the gel. Once the RNA interacts with the buffer, it gets transferred to the membrane. A stack of paper towels and weight kept on the membrane enhances the imprint of the bands on the membrane. Now the RNA molecules present on the membrane exactly resemble as they were on the gel.

Step 4: Hybridization with the labeled probe:

Exposure of the membrane to the probe ensures hybridization of RNA molecules. Northern blotting procedure involves probes composed of complementary sequences of RNA. It consists of at least 25 complementary bases. Northern blotting uses cDNA as a probe. Radioactive labeling of the probe includes a radioactive isotope P³². Alternative labeling involves non-radioactive techniques such as chemiluminescence labeling. The chemiluminescence technology involves the breakdown of the chemiluminescence substrates through enzymes such as alkaline phosphatase or horseradish peroxidase. The substrates produce a detectable emission of light.

Step 5: The procedure of chemiluminescent labeling:

The probe (cDNA) gets attached to the enzyme alkaline phosphatase or horseradish peroxidase. Alternatively, labeling of the probe may involve a ligand. The ligand gets attached to the enzyme. Determination of the efficiency of the hybridization includes ionic strength, viscosity, duplex length, and base composition. Non-hybridized probes get removed by washing the membrane filter gently.

Step 6: Exposure to the X-ray film:

An X-ray film detects the signals generated with the chemiluminescent-labeled probes hybridized to RNA. A quick and sensitive signal generation occurs through chemiluminescent labels.

Step 7: Quantification of RNA through densitometry:

The process of densitometry involves a quantitative measurement of the optical density in the light-sensitive material.

Following is the summary of the northern blotting procedure:

1.     Isolation of RNA from a tissue sample.
2.     Loading the samples and the markers in the electrophoretic wells.

3.     Fragments get separated based on their sizes. Molecules with larger sizes are near the wells. Molecules with smaller sizes move faster. The fragments appear in the form of bands.

4.     Exposure of the gel to a buffer solution and a membrane filter.

5.     RNA gets transferred from the gel to the membrane.

6.     RNA hybridization using probes.

7.     Signal detection

8.     RNA quantification

Applications of northern blotting:

Gene expression study includes the pattern of gene expression in the tissues, organs and developmental stages. Northern blotting applies in studying the overexpression of oncogenes, the upregulation or downregulation of oncogenes and tumor-suppressor genes. It may help to find a gene function. Northern blotting helps to check the cloned DNA. Especially it checks the cloned cDNA since the technique uses cDNA as a probe to detect specific RNA. Analysis of micro RNAs becomes easy with this technique. A high resolution northern blotting monitors RNA expression. Micro RNAs are short and non-coding regulatory molecules. Post-transcriptional regulation of genes involves the role of micro RNA. The technique is readily available. Hence it is used in micro RNA analysis. The probes used in the technique are known as locked nucleic acid modified oligonucleotide probes. They are extremely sensitive and specific in detecting mature micro RNAs.

Immuno-northern blotting detects RNA modification through antibodies. The RNA gets separated through electrophoresis and transferred to the membrane. Immuno-blotting involves antibodies. It reveals antibody cross-reactions, characterization of antibodies, and modified nucleosides. It is a highly specific technique. The northern blotting technique also characterizes the RNA interference reagents. The comparison of the data derived from deep sequencing of micro RNAs with endogenous and exogenous RNAs is possible with the northern blotting technique. A gene silencing phenomenon is known as RNAi or RNA interference. The double-stranded RNAs get processed into small interfering RNAs (siRNA). A siRNA acts like a guide and enables cleavage of a homologous RNA. It occurs mainly in the RNA induced silencing complex (RISK). High-resolution northern blotting efficiently detects the length heterogeneity of the RNAi technology reagents.

A blot-base is an online database used to publish northern blots. It is a database used in genome sequencing, determination, and the protein structure.

Reverse northern blotting:

It is a variant of northern blotting technique. In this, the DNA fragments get hybridized with the RNA probes labeled radioactively. It enables gene expression profiling.

Virtual northern blotting:

It involves a comparison of relative amounts of transcripts in different tissues. The comparison is between small quantities of total RNA and full-length cDNA.

Advantages and disadvantages of northern blotting:

Detection of RNA size and observation of alternate spliced products become easy with this method. The technique is applicable for quantitative as well as qualitative analysis. It has a high specificity. With so many advantages, there are disadvantages too. As compared with RT-PCR, northern blotting has a low sensitivity. Analyzing thousands of genes at a time is not possible. The RNases are always ready to degrade the sample. Hence the technique always requires RNase inhibitors and proper sterilization of glassware. Chemicals used in northern blotting may be risky.

References:
[1] Molecular Biology Techniques: An Intensive Laboratory Course, Walt Ream, Katharine G. Field

[2] Ana Techniques in Biotechnology, Goutam Bhowmik

[3] Gene Cloning and DNA Analysis, T.A. Brown
[4] Molecular Plant Biology: A Practical Approach, Volume 2, Philip M. Gilmartin, P. M. Gilmartin, Dr. Chris Bowler

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