Northern Blot Technique Notes

Northern Blot Technique

Formaldehyde is added to RNA samples for denaturation. This is important because:
- RNA is typically single-stranded but can form secondary structures due to self-complementary sequences, leading to intrachain base pairing and folding.
- Formaldehyde contains a carbonyl group that reacts with amino or imino groups of nucleobases to form Schiff bases.
- These covalent adducts prevent normal base pairing and maintain the RNA in a denatured state.

RNAs are separated by gel electrophoresis.
Loading buffer is added to the samples as a tracking dye to monitor the separation progress.
Agarose formaldehyde gel electrophoresis is commonly used.
An RNA ladder (molecular weight size marker) is used to determine the size of RNA molecules.
- The RNA ladder is added into a well at one end of the gel.
- RNA samples are then loaded into wells.
- An electric current is applied, causing the RNA molecules to migrate through the gel.
RNA molecules are negatively charged due to their phosphate backbone; therefore, they migrate towards the positively charged anode.
Smaller RNA molecules move faster than larger ones because all RNA molecules have the same charge per mass.
Formaldehyde must be present in the agarose gel to maintain the RNA in a denatured state since RNA-formaldehyde adducts are unstable.

After electrophoresis, the RNA molecules in the gel are stained with an intercalating dye, such as ethidium bromide.
Under UV light, RNA molecules appear as bands, each representing a group of same-sized RNA molecules.

RNA molecules are transferred from the gel to a membrane.
This process involves a transfer buffer, a solid support, and blotting paper, which acts as a wick for the transfer solution.
The wick is placed over the solid support in the transfer reservoir with its ends 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), pre-wetted with the transfer solution, is placed on top of the gel.
Pre-wetted pieces of extra thick blotting paper are placed on top of the membrane.
Exposed areas of the wick are covered with plastic wrap to prevent buffer bypass.
A dry stack of paper towels is placed on top of the membrane and gel, and a glass plate with a weight is applied to maintain tight contact.
Buffer transfer occurs by capillary action, moving RNA from the gel onto the membrane.
Ion exchange interactions bind the RNA to the membrane due to the negative charge of the RNA and the positive charge of the membrane.
The transfer process usually proceeds overnight.

After transfer, the blotting material and membrane are carefully removed from the gel.
The membrane is briefly rinsed to remove any agarose.
It is then exposed to ultraviolet (UV) radiation to permanently attach the transferred RNA.

The membrane is placed in a bottle containing a pre-hybridization solution to reduce non-specific hybridization.
The bottle is incubated in a hybridization oven at 42 degrees Celsius for 2 hours.
The pre-hybridization solution is removed, and a hybridization buffer is added.
Labeled probes are added to the hybridization solution.
Commonly, cDNA is created with labeled primers for the RNA sequence of interest to serve as the probe and can be radioactively or fluorescently labeled.
The bottle is incubated overnight in the hybridization oven at 42 degrees Celsius.
DNA contains a large quantity of phosphorus in the phosphodiester linkages between nucleotides.
DNA can be tracked by replacing its non-radioactive phosphorus with radioactive phosphorus-32 (^{32}P).
The radioactively labeled DNA probes hybridize to their complementary sequences in the RNA molecules.

After hybridization, the hybridization solution is removed.
A wash buffer is added, and the membrane is incubated at 52 degrees Celsius for 30 minutes.
The washing process is repeated three times to remove unbound and weakly-bound probe.

An autoradiography method is used to identify the location of radioactively labeled RNA on the membrane.
The membrane is placed inside a light-proof cassette box with an X-ray film laid over the top.
The cassette is closed and left for several hours to several days.
The radioisotope labeled RNA exposes the film, which, upon development, shows a pattern of black bands indicating the positions of labeled RNA in the blot membrane.
This identification is used to determine which RNA molecule is present in each sample.