DETECTION AND ANALYSIS OF NUCLEIC ACIDS - A Guide to Methods in the Biomedical Sciences - page 36

Northern blotting

This is a method designed to identify individual RNA that has been
fractionated on agarose gels and transferred to a membrane for probing.
It is therefore analogous to Southern blotting for DNA. The only major
difference is that because RNA can form secondary structures, it must
be resolved on gels under denaturing conditions. Common denaturants
include formaldehyde and glyoxal.
Northern blotting got its name from its developers, Alwine and Stark
(17), who called it “Northern” blotting as a joke (after “Southern” blotting),
but the name has stuck, and other blotting methods have since used
compass directions as descriptions as well. As for Southern blotting,
the probes used are generally DNA probes (cDNA probes, or dou-
ble stranded genomic DNA fragments that are denatured before use).
Northern blotting is commonly used to identify a mRNA transcript in
either total cellular RNA or in enriched mRNA. mRNA can be puri-
fied using oligo dT cellulose and then fractionated on agarose gels
in order to detect rarer transcripts, since most cellular RNA is riboso-
mal and transfer RNA (tRNA). Northern blots can provide quantitative
measures of steady state RNA levels, especially comparative measure-
ments of the relative abundance of RNA in different cell types. How-
ever, Northern blotting is not as quantitative as ribonuclease protection
assays.

Ribonuclease (RNase) protection assay (RPA)

RPA is used to quantitate steady state levels of RNA, to identify
the or ends of mRNA, and to characterize the splice junctions
of the primary RNA transcripts that are processed into mRNA. The
probes used for RPA are RNA probes that are generated by RNA poly-
merase from bacteriophage promoters, including SP6 (from Salmonella
typhimurium) and T3 or T7 (from E. coli The DNA to be used for the

probe is cloned into a vector downstream of a bacteriophage promoter,
and the RNA polymerase is used to produce an RNA probe (usually con-
taining radioactive precursors) complementary to mRNA. Once the RNA
probe is hybridized to RNA, RNase is used to remove the free probe and
any single stranded segments of hybridizing RNA. The probe will be di-
gested at any point where the probe and RNA do not hybridize. This can
be the end of the RNA, or regions marking exon-intron boundaries.
The remaining RNA which was “protected” by annealing to the RNA of
interest is then fractionated on a sequencing gel, and the exact size and
abundance of the probe remaining can therefore be determined. The
size of the protected fragment reveals the start site or domain boundary,

).