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

                                  © Copyright, 2018 All Rights Reserved.

Southern blotting technique

The gel electrophoresis technique separates the DNA fragments based on their sizes. Smaller molecules move faster than the larger molecules. A basic question arises as to why separate these fragments? The reason is very simple. Separation of fragments based on the sizes helps to obtain specific fragments from the gel instead of getting the entire genomic DNA. Hybridization involves finding the location of a gene or its product using a nucleic acid probe. Most of the times, the probes are small single-stranded DNA molecules. Determination of complementary sequences utilizes hybridization techniques. Hence, the probes bind only to the complementary sequences. Isolated bands from electrophoretic technique determine an efficient mapping of DNA sequences or gene detection. Blotting technique facilitates hybridization. The process involves the transfer of bands to a nitrocellulose membrane. There are three types of blotting procedures depending on the type of the molecule. Southern blotting is used to blot the DNA. Northern blotting is used to blot the RNA. Western blot involves the transfer of protein bands. E.M. Southern derived the southern blotting method for the first time.

Image: Southern blotting

Analyzing the sequences using southern blotting:

Step 1: Treatment with a restriction enzyme:

The DNA undergoes a treatment with a restriction enzyme. The enzymes cleave the DNA into various fragments. The process of cleaving the DNA to obtain fragments is known as restriction digestion. The fragments obtained from restriction digestion are known as restriction digests. Restriction enzymes are known as molecular scissors as they cut the DNA at specific sites known as the restriction sites.

Step 2: Separation of the fragments through gel electrophoresis:

The main aim of restriction digestion involves studying the DNA in bits and pieces and picking up the piece of interest for analyzing. Electrophoresis does the work of separating the fragments as per the sizes. Not only DNA but also RNA can be separated. The principle of electrophoresis is simple. The DNA is a negatively charged molecule. It migrates toward the positive electrode. A positively charged molecule moves toward the negative electrode. The shape of the molecule, the charge, and the molecular length determine the rate of migration. Only one criterion of gel electrophoresis involves molecular length. The composition of the gel mainly constitutes agarose, which is nothing but a network of pores through which DNA molecules travel. Molecules of different lengths form bands on the gel.

Step 3: Staining the DNA

Staining the DNA involves ethidium bromide. This chemical is a carcinogen and neurotoxic. Use it with precaution. Staining with the ethidium bromide helps in visualizing the bands under ultraviolet light. Ethidium bromide intercalates with the DNA.

Step 4: Transferring the gel to a membrane filter:

The gel consisting of DNA fragments gets transferred to the membrane filter. Following description is about the apparatus. Firstly, a buffer solution poured into a tray serves as an alkaline medium. Soaking the gel in the buffer solution denatures the DNA into single strands. Next step involves neutralization of the gel and placing the blotting paper. The ends of the paper act as a wick that takes up the buffer solution until the gel. The membrane filter covers the gel. The next step involves placing the paper towels and weight on the filter. Due to the blotting action, the buffer solution travels through the gel onto the membrane filter. The DNA fragments get picked up by the buffer solution and get transferred to the membrane.

Step 5: Hybridization with the probes:

The probes may or may not be radioactively labeled. The process involves the addition of the probe to the membrane filter so that the DNA present on the filter gets hybridized with the probe.

Step 6: Autoradiography:

Permanent fixation of the DNA on the membrane involves heating at 80⁰C for 2-3 hours. Now, the DNA gets completely hybridized with a labeled DNA probe. The probe forms a complementary base pair with the homologous sequence on the DNA fragment. Unbound probes are removed by carefully washing the membrane. Autoradiography technique involves an X-ray sensitive photographic film. Exposing the membrane filter to the X-ray sensitive photographic film determines the labeled molecules.

In summary, the southern blotting technique involves restriction digestion, gel electrophoresis, probing and autoradiography.

Applications of the southern blotting:

1.     SNP analysis:

Single base pair changes constitute single nucleotide polymorphisms. Southern blotting efficiently determines SNP alleles. The initial step involves the isolation of genomic DNA and digestion with the restriction enzymes. The electrophoretic techniques separate the fragments based on their sizes in kilobases. The action of the blotting paper helps in transferring the DNA present on the gel to the membrane filter placed on top of the gel. A stack of paper towels and weight kept above the membrane helps in keeping the membrane fixed at one place. Hybridization with the probe enables complementary base pairing. The visualization of the bands under an X-ray sensitive photographic film gives a clear picture of the DNA. Southern blotting helps in detecting homozygotes and heterozygotes. Comparison of bands becomes easy with the southern blotting.

2.     DNA molecular testing with ASOs:

It includes allele-specific oligonucleotide hybridization or short oligonucleotides complementary to SNP alleles. The oligonucleotides mixed with DNA get hybridized. ASO hybridization also involves Southern blotting. The ASOs labeled radioactively get hybridized with the DNA immobilized on the membrane filter. Analysis of the resulting autoradiograms helps in detecting gene mutations.

3.     RFLP analysis:

RFLP analysis includes detection of genetic disorders such as PKU, sickle cell anemia, and many others. Restriction fragment length polymorphisms or RFLP analysis exploits homologous DNA variations.

4.     Zoo blot:

A blot consisting of DNA from a variety of organisms is known as a zoo blot. The digestion of DNA obtained from organisms such as chicken or a hamster with the restriction enzymes gives fragments of different lengths. The analysis of these fragments includes southern blotting.

5.     DNA typing or DNA fingerprinting:

Digestion of DNA with endonucleases giving fragments, later on electrophoresed, give banding patterns on the gel. The southern blot of the probe gets further probed with the VNTR-specific probes in the DNA fingerprinting technique. Applications of DNA fingerprinting include paternity and maternity testing, studying mitochondrial inheritance, and crime scene investigation.

6.     DNA microarray involving southern blotting:

Southern hybridization with DNA microarray includes unlabelled DNA probes targeting label-free DNA molecules. However, DNA microarray uses a chip or a probe array. The method uses fluorescence dyes or cyanine dyes.

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

                                       © Copyright, 2018 All Rights Reserved.