This classical #technique was developed by James Alwine, David Kemp and George Stark in 1977 at #Stanford University. It has multiple applications in #MolecularBiology research. Lets get into it!
Principle
The principle here is detection of RNA by radioactive DNA probes by means of hybridization.
Protocol
Often total cellular #RNA from homogenized tissue samples are run on denaturing #AgaroseGel supplemented with #formaldehyde.
The gel bands are then transferred to a #NitroclluloseMembrane through a capillary or vacuum blotting system. A nylon membrane with a positive charge is most effective for use here as negatively charged RNA has high affinity for them. The transfer buffer often contains #Formamide because it lowers the annealing temperature of the probe-DNA interaction, thus eliminating the need for high temperature, which may cause RNA degradation.
Once the RNA is transferred on the membrane, it is immobilized through #covalent linkage to the membrane by #UVLight or by hot #VacuumDrying.
The membrane is then moistened with a #probe solution of #P32-labelled DNA single strands complementary in base sequence to the target RNA. It is then incubated for several hours at an appropriate renaturation temperature to hybridize the RNA strands with the probe DNA strands.
The unbound probe is washed away from the membrane, leaving only the bands of radioactive DNA-RNA hybrid duplex strands on it.
The membrane is next #autoradiographed to bands on the autoradiograph film, corresponding to respective positions of the #radioactive hybrid duplexes.
Merits
Observation and identification of alternate splice products or #SpliceVariants.
The nylon membrane can be stored and reprobed (upto 5 times) for years after blotting without a significant loss of the target RNA.
This method has high #sensitivity.
Demerits
#Genes expressed in specific #developmental time points cannot be detected surely at all times.
Members of #multigene families cannot be ascertained, compromising on #specificity.
Compared to #RTPCR it has low sensitivity.
Degradation of RNA is a concern
The chemicals used in the method can be a risk to the researcher. #Formaldehyde, #radioactive probes, #EthidiumBromide, #DEPC, #UV light are all harmful under certain exposures.
Applications
Determination of particular #gene expression levels.
Determination of number of genes present in a #DNA fragment and also the size of each #CodingRegion.
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