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Posted on Thu, Oct 11, 2018

Northern Blot Protocol

The northern blot is a technique used to study gene expression via mRNA transcripts. The northern blot was named after the southern blot, which was developed to study DNA. The two techniques are the same except that the northern blot is used to detect RNA while the southern blot is used to detect DNA. The northern blot protocol, in brief, involves gel electrophoresis to separate mRNA by size, a blotting step to transfer the separated mRNA to a membrane, and a probe hybridization step to identify the mRNA sequence of interest. Even with the advent of powerful RNA analysis techniques such as RT-qPCR and sequencing, the northern blot is still useful for comparing gene expression between samples. The northern blot protocol is relatively inexpensive, and makes it easy to visualize the results on a single membrane.  

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Northern blot protocol:

  1. Extraction of RNA:

  • There are many RNA extraction kits commercially available, but they all involve cell lysis, inhibition of RNAases, removal of proteins and other contaminants, and recovery of RNA

2. Isolation of mRNA:

  • Oligo dT cellulose chromatography can be used to isolate only mRNA with a polyA tail. The poly A tail is the final step of mRNA production in the nucleus. The tail enables nuclear export, translation, and stability of mRNA. In Oligo dT cellulose chromatography, oligos complementary to the poly A tail are covalently attached to a resin column. When the sample is applied to the column the mRNA with the poly A tail will hybridize to the oligo probe and be retained on the column. Then, the elution buffer is applied to disrupt hybridization and recover the mRNA.


3. Gel electrophoresis to separate mRNA by size:

  • Agarose gels containing formaldehyde were traditionally used to denature RNA*. The formaldehyde reacts with the imine and amine groups on the nucleic acids, which disrupts the hydrogen bonding between bases and disrupts the secondary structure of the RNA. It is important to disrupt the secondary structure because the RNA must be extended to allow proper binding of probe for identification.


4. Transfer of RNA to blotting membrane:

  • The transfer is necessary because the probes can’t enter into the gel matrix. Therefore, the RNA must be transferred to a membrane where they can be accessed by the probes
  • Transfer is accomplished via a capillary (overnight) or vacuum (15-60 minutes) blotting system.
  • The blotting membrane is positively charged to attract the negatively charged RNA. Nylon is a commonly used membrane.


5. Immobilization of RNA to the blotting membrane:

  • Covalently attached to the membrane by the application of UV light or heat.


6. Application of Probe:

  • Probes have a minimum of 25 bases that are complimentary to the mRNA sequence of interest.
  • Excess probe is washed off


7. Probe visualization:

  • Radioactive isotopes were traditionally used, but have been replaced in favor of safer detection methods.
  • Chemiluminescence is commonly used in the modern northern blot protocol


*Glyoxal, an alternative to formaldehyde, is now available is many commercial northern blot kits. Formaldehyde is a suspected human carcinogen, and has been shown to cause squamous cell carcinoma in the nasal passages and trachea in rats exposed to inhalation. Formaldehyde denatures large RNA molecules by forming single point adducts with the amine or imine molecules on base pairs and disrupting normal hydrogen bonding. These linear adducts are unstable and will decompose if formaldehyde is not present in the electrophoresis gel. This means that gels must be poured and run in a fume hood in order to reduce the human exposure risk. Glyoxal is a diformyl molecule that contains two carbonyl groups, which means that it can react with neighboring amines and imines at the same time on a base pair and form a more stable cyclic adduct with two points of contact. The stability of glyoxal denaturing is attractive because it eliminates the need to pour a denaturing gel. The glyoxal can be applied to the RNA prior to gel electrophoresis, and the gel does not need to contain glyoxal or formaldehyde.

 

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