Northern Blot Technique: A Comprehensive Summary

Northern Blot: Revealing RNA Identity, Size, and Abundance

Northern blotting is a molecular biology technique used to study gene expression by detecting specific RNA molecules within a sample. It provides information about RNA identity, size, and abundance.

1. RNA Sample Preparation

  • Formaldehyde is added to RNA samples to denature the RNA molecules.
  • RNA, being a single-stranded nucleic acid, often forms secondary structures due to self-complementary sequences.
  • Intrachain base pairing results in folding, leading to complex structural forms.
  • Formaldehyde's carbonyl group reacts with amino or imino groups of nucleobases, forming Schiff bases.
  • These covalent adducts prevent normal base pairing, maintaining the RNA in a denatured state.

2. Gel Electrophoresis for RNA Separation

  • A loading buffer is added to RNA samples, serving as a tracking dye to monitor the separation progress.
  • Agarose gel electrophoresis is commonly used for RNA molecule separation.
  • An RNA ladder (molecular weight size marker) is used to determine RNA molecule sizes.
  • The RNA ladder is loaded into a well at one end of the gel.
  • RNA samples are loaded into other wells.
  • An electric current is applied to pull the samples through the gel.
  • Based on charge and size, RNA molecules migrate at different speeds, enabling separation.
  • The negatively charged phosphate backbone of RNA causes migration toward the positively charged anode in an electric field.
  • Smaller molecules move faster than larger ones due to uniform charge-to-mass ratio.
  • Formaldehyde must be present in the agarose gel to maintain RNA denaturation due to the instability of RNA-formaldehyde adducts.

3. Visualization of RNA Molecules

  • After electrophoresis, RNA molecules are stained using an intercalating dye (e.g., ethidium bromide).
  • Under UV light, RNA bands become visible, each band representing same-sized RNA molecules.

4. Northern Blotting: Transfer to Membrane

  • A transfer buffer, solid support, and blotting paper wick are used to transfer RNA molecules from the gel to a membrane.
  • The wick is placed over the solid support in the transfer reservoir with ends submerged in the transfer buffer.
  • Extra-thick blotting paper is placed on top of the wick and wetted with the transfer solution.
  • The gel is placed on the wetted wicking paper.
  • A nylon membrane (same size as the gel) is pre-wetted with the transfer solution and placed on top of the gel.
  • Pre-wetted, extra-thick blotting paper is placed on top of the membrane.
  • Exposed wick areas are covered with plastic wrap to prevent buffer bypass.
  • A dry stack of paper towels is placed on top of the membrane and gel, with a glass plate and weight on top to maintain tight contact.
  • Capillary action drives buffer transfer from high to low water potential areas, moving RNA from the gel onto the membrane.
  • Ion exchange interactions bind the negatively charged RNA to the positively charged membrane.

5. RNA Fixation and Hybridization

  • The transfer process occurs overnight.
  • Blotting material and membrane are carefully removed from the gel.
  • The membrane is briefly rinsed to remove any adhering agarose.
  • UV radiation permanently attaches the transferred RNA to the membrane.
  • Hybridization with radiolabeled probes is performed.
  • The membrane is placed in a bottle with a pre-hybridization solution to reduce nonspecific probe hybridization.
  • The bottle is incubated in a hybridization oven at 42^\circ C for two hours.
  • The pre-hybridization solution is removed, and a hybridization buffer is added.
  • Labeled probes are added to the hybridization solution.
  • cDNA, created with labeled primers for the RNA sequence of interest, serves as the probe.
  • Probes can be radioactively or fluorescently labeled.
  • The bottle is incubated overnight in the hybridization oven at 42^\circ C.
  • DNA contains phosphorus in its phosphodiester linkages, which can be tracked by replacing non-radioactive phosphorus with radioactive phosphorus-32 (^{32}P).
  • Radioactively labeled DNA probes hybridize to complementary sequences in the RNA molecules.

6. Post-Hybridization and Autoradiography

  • After hybridization, the hybridization solution is removed.
  • A wash buffer is added, and the membrane is incubated at 52^\circ C for 30 minutes.
  • The washing process is repeated three times to remove unbound probe.
  • Autoradiography identifies the location of radiolabeled RNA on the membrane.
  • The blot filter is placed inside a light-proof cassette box, and an X-ray film is laid over it.
  • The cassette is closed and left for several hours to several days.
  • Radioisotope-labeled RNA exposes the film, which, upon development, shows black bands indicating the positions of labeled RNA in the blot membrane.
  • This identification determines which RNA molecule is present in each sample.