Biochemical and Molecular Biological Assessments of Intracerebral Hemorrhage - Animal Models of Acute Neurological Injuries: Injury and Mechanistic Assessments - page 638

2.3. Notes

1. The protocol described here is an example of the many possi-
bilities for immunoassay. Many different variations are possible.
Although we have given a protocol using a biotinylated sec-
ondary antibody, the primary antibody can be biotinylated and
the avidin-enzyme probe can then be used immediately after-
ward. Of course, other avidin-conjugated enzymes, other
probes (fl uorescent, chemiluminescent, radioactive, etc.), or
avidin complexed with the biotinylated probes can be substi-
tuted for the avidin-conjugated peroxidase given as an example
in this section.
2. The optimal concentration of antibody for coating plates varies
for any given preparation and should be determined empirically
for each system. Likewise, other components of the assay system
should be pretested one at a time in model experiments.

3. The Polymerase
Chain Reaction

The polymerase chain reaction (PCR) is a cardinal laboratory tech-
nology in molecular biology, and represents an in vitro method of
nucleic acid synthesis by which a particular segment of DNA is
specifi cally replicated ( 6 ). Real-time PCR is a form of the PCR that
maximizes the potential of the technique.

3.1. Principles of PCR

PCR involves two oligonucleotide primers—small pieces of DNA
complementary to the gene of interest—that fl ank the DNA frag-
ment to be amplifi ed and repeated cycles of heat denaturation of
the DNA, annealing of the primers to their complementary
sequences, and extension of the annealed primers with DNA poly-
merase. These primers hybridize to opposite strands of the target
sequence and are oriented so that DNA synthesis by the polymerase
proceeds across the region between the primers. Since the exten-
sion products themselves are also complementary to and capable of
binding primers, successive cycles of amplifi cation essentially dou-
ble the amount of the target DNA synthesized in the previous
cycle. The result is an exponential accumulation of the specifi c tar-
get fragment ( 7 ).
In order for PCR amplifi cation to be successful, the nucleic
acid target molecule must be readily accessible to primers and DNA
polymerase and free from inhibitory concentrations of containing
proteins, lipids, carbohydrates, and salts. This necessitates that the
DNA or RNA target molecule is fi rst liberated and purifi ed from
both the cellular environment in which it resides and the chaper-
one proteins which may accompany it. Many commercially avail-
able kits for DNA and/or RNA purifi cation are currently available,
employing enzymatic, chemical, or mechanical extraction technique.