After an animal has been injected with a radioactive precursor of RNA,
some of it will be incorporated into DNA as well as into RNA (remember
that the precursors of RNA lack specificity), and part of the precursor
will be broken down into smaller molecules. The injected animal can be
sacrificed, and an organ or another tissue, for instance, the liver, can
be removed. Then the liver is homogenized, that is, ground to a pulp
with a modern version of the mortar and pestle. The homogenate (pulp) is
treated with cold (weak) acid. Proteins and nucleic acids are insoluble
in cold acids and therefore precipitate to the bottom of the test tube.
All molecules that are soluble in a cold acid are left in the
supernatant (the remaining liquid); among these are small molecules,
like those of the RNA precursor. The precipitate (the solid material
that settles to the bottom), now containing proteins and nucleic acids,
is then treated with a strong alkali, for instance, sodium hydroxide.
Alkali will digest RNA into smaller molecules but does not affect DNA.
If we now add acid to the solution, DNA, being insoluble in acid, will
precipitate again; RNA, having been broken down into small molecules,
will remain in the supernatant. DNA can then be extracted from the
precipitate by boiling in strong acid. Proteins from the tissue remain
in the final residue.
We have now fractionated the tissue into four portions: the acid-soluble
fraction (containing small molecules), RNA, DNA, and proteins. (The
cell’s lipids and sugars come out during alcohol rinses between the weak
acid and the alkali steps.) Chemical analysis allows us to measure
precisely the amount of RNA or DNA in its respective fraction and
therefore in the tissue or organ. The amount of radioactivity in the RNA
fraction can then be determined by a technique known as liquid
scintillation counting.