Northern Blotting
A northern blot is a laboratory technique used to identify particular RNA molecules among a mixture of RNA. Northern blotting can be used to analyze a sample of RNA from a particular tissue or cell key to determine the RNA expression of particular genes.
Northern blotting was the first procedure developed for evaluating the molecular size and abundance of selective RNAs in a mixture of RNAs or nucleic acids.
This process trusts the process of nucleic acid hybridization in between a recognized nucleic acid probe and the complementary sequence in a population of RNAs.
The northern blot method was developed in 1977 by James Alwin, David Kemp, and George Stark at Stanford University, with contributions from Gerhard Heinrich. Northern blotting takes its name from its similarity to the first blotting technique, the Southern blot.
Principle of Northern blotting
Northern blot first uses denaturing gel to separate RNA according to the size. The RNA is then transferred to a nylon membrane while keeping the exact same distribution in the gel. After fixing the RNA to the membrane, a labeled probe complementary to the gene of interest is then added to hybridize to the debilitated RNA.
The nonspecifically bounded probes are then washed away. The solid membrane with a probe specifically bound to RNA of interest is then dried, exposed, and evaluated.
Given that northern blot uses size-dependent separation, this strategy can not only figure out the abundance but likewise the sizes of a transcript of interest.
Equipment required
- Agarose gel rig
- Power supply
- Vacuum gel transfer system
- Nylon membrane
- Phosphor screen
- Phosphor screen scanning equipment
- Hybridization oven
- Hybridization bottles
Steps involved in Northern blotting
RNA is isolated from several samples such as skin, hair, the cornea of eyes, sperm/ egg cells.
Samples are loaded in agarose gel.
The RNA sequence is separated in the electrophoresis system an agarose gel is used for the function of the nucleic acid separation.
Now the separated RNA sequence is transferred to the nylon membrane. This is done by two systems capillary action and ionic interaction.
The transfer is done by keeping the gel in the sandwich arrangement. First, the agarose gel is placed on the bottom of the stack, followed by the blotting membrane. On top of these paper towels, a mild weight (glass plate) is positioned. The entire setup is kept in a beaker including a transfer buffer.
RNA transferred to the nylon membrane is then fixed using UV radiation.
The fixed nylon membrane is then combined with probes. The probes are specifically developed for the gene of interest so that they will hybridize with RNA series on the blot corresponding to the sequence of interest.
The blot membrane is washed to get rid of unwanted probes.
The labeled probe is detected by chemiluminescence or autoradiography. The outcome will be dark bands in x-ray film.
Advantages and Uses
- Northern blotting can be utilized to determine novel splice versions, pre-processed RNAs, and non-coding RNAs, along with their relative abundances.
- This technique reveals the identity, number, activity, and size of the particular gene.
- This blotting technique can also be utilized for the development of a tissue or organism.
- In different phases of differentiation and morphogenesis, the abundance of RNA modifications and can be identified using this strategy.
- It also helps in the recognition of unusual, diseased, or infected conditions at the molecular level.
Disadvantages
- Northern blotting is relatively less sensitive than RTPCR.
- Detection of multiple probes is a problem.
- Non-specific matching of the probe to RNA can take place.
- RNA is slightly degraded by RNase due to contamination by water, contaminated hands, or tips and equipment can negatively affect the quantitation.
MCQs
- Which laboratory technique is used to identify particular RNA molecules among a mixture of RNA?
- A) Southern Blotting
- B) Northern Blotting
- C) Western Blotting
- D) Eastern Blotting
- Answer: B) Northern Blotting
- Who developed the Northern blotting technique?
- A) James Alwin
- B) David Kemp
- C) George Stark
- D) All of the above
- Answer: D) All of the above
- What is the principle behind Northern blotting?
- A) DNA hybridization
- B) Protein isolation
- C) Nucleic acid hybridization
- D) Gel electrophoresis
- Answer: C) Nucleic acid hybridization
- What is the purpose of transferring RNA to a nylon membrane in Northern blotting?
- A) To increase RNA concentration
- B) To facilitate gel electrophoresis
- C) To perform hybridization with probes
- D) To degrade RNA
- Answer: C) To perform hybridization with probes
- Which equipment is NOT required for Northern blotting?
- A) Agarose gel rig
- B) Power supply
- C) PCR machine
- D) Hybridization oven
- Answer: C) PCR machine
- Which step follows RNA isolation in the Northern blotting process?
- A) Gel electrophoresis
- B) Hybridization with probes
- C) Fixation using UV radiation
- D) Washing away nonspecifically bounded probes
- Answer: A) Gel electrophoresis
- What is the main advantage of Northern blotting?
- A) High sensitivity
- B) Low cost
- C) Determination of RNA abundance and size
- D) Detection of multiple probes simultaneously
- Answer: C) Determination of RNA abundance and size
- What is NOT a use of Northern blotting?
- A) Identification of splice variants
- B) Determination of protein structure
- C) Recognition of unusual disease conditions
- D) Study of RNA modifications
- Answer: B) Determination of protein structure
- What is a disadvantage of Northern blotting compared to RT-PCR?
- A) Higher sensitivity
- B) Detection of multiple probes
- C) Less susceptibility to nonspecific matching
- D) Relatively less sensitive
- Answer: D) Relatively less sensitive
- How is RNA transferred to a nylon membrane in Northern blotting?
- A) Via PCR
- B) Via capillary action and ionic interaction
- C) Via gel electrophoresis
- D) Via hybridization
- Answer: B) Via capillary action and ionic interaction
- What does the term “blotting” refer to in Northern blotting?
- A) Blurring of RNA bands
- B) Blotting out irrelevant information
- C) Transfer of RNA to a membrane
- D) None of the above
- Answer: C) Transfer of RNA to a membrane
- Which technique is used to detect labeled probes in Northern blotting?
- A) Chemiluminescence
- B) Fluorescence
- C) Absorbance spectroscopy
- D) Nuclear magnetic resonance
- Answer: A) Chemiluminescence
- What can negatively affect RNA quantitation in Northern blotting?
- A) RNase contamination
- B) Gel electrophoresis
- C) Hybridization efficiency
- D) UV radiation
- Answer: A) RNase contamination
- What is the purpose of fixing RNA to a nylon membrane in Northern blotting?
- A) To degrade RNA
- B) To facilitate gel electrophoresis
- C) To perform hybridization with probes
- D) To increase RNA concentration
- Answer: B) To facilitate gel electrophoresis
- Which technique is NOT used in the Northern blotting process?
- A) Gel electrophoresis
- B) PCR amplification
- C) Hybridization
- D) Autoradiography
- Answer: B) PCR amplification
- What does RTPCR stand for?
- A) Reverse Transcription Polymerase Chain Reaction
- B) Rapid Thermal Processing of PCR
- C) Real-Time Polymerase Chain Reaction
- D) Random Transcription Polymerase Chain Reaction
- Answer: A) Reverse Transcription Polymerase Chain Reaction
- What is the significance of the term “Northern” in Northern blotting?
- A) Named after the region where it was first developed
- B) Named after the direction of the transfer of RNA
- C) Named after the first researcher who used it
- D) Named after the type of RNA used
- Answer: A) Named after the region where it was first developed
- What type of RNA is analyzed in Northern blotting?
- A) Ribosomal RNA
- B) Messenger RNA
- C) Transfer RNA
- D) All of the above
- Answer: B) Messenger RNA
- Which technique is NOT commonly used for RNA analysis?
- A) Western blotting
- B) Southern blotting
- C) Northern blotting
- D) Eastern blotting
- Answer: A) Western blotting
Summary of Northern Blotting:
Introduction: Northern blotting is a vital laboratory technique used to identify specific RNA molecules within a mixture, enabling the analysis of RNA expression in tissues or cells.
Principle: The technique relies on nucleic acid hybridization, involving the separation of RNA by size, transfer to a nylon membrane, and hybridization with a labeled probe specific to the target gene.
Procedure: The steps of Northern blotting include RNA isolation, gel electrophoresis, membrane transfer, fixation, hybridization with probes, washing, and detection of labeled probes.
Advantages and Uses: Northern blotting facilitates the determination of RNA abundance, splice variants, and non-coding RNAs. It aids in identifying gene activity, size, and studying RNA modifications, tissue development, and disease conditions.
Disadvantages: Despite its utility, Northern blotting has limitations such as lower sensitivity compared to RTPCR, potential probe detection issues, non-specific matching of probes to RNA, and susceptibility to RNA degradation.
In conclusion, Northern blotting is a valuable tool in molecular biology, offering insights into RNA expression and function, albeit with certain drawbacks that researchers must consider.