Characterization of RNA - Animal Models of Acute Neurological Injuries: Injury and Mechanistic Assessments - page 147

resulting cDNA is amplifi ed using traditional or real-time PCR. If
a conventional PCR is used, the PCR product is detected using
agarose gel electrophoresis and EtBr (or other nucleic acid staining);
however, more time-consuming and important limitations (low
sensitivity and reliability) can be expected when compared to real-
time PCR techniques ( 54 ).
Real-time PCR allows for the detection of PCR products via
the generation of a fl uorescent signal. There are four major kinds
of fl uorescent reporters used in RT-PCR: SYBR Green I, TaqMan
Probes, Molecular Beacons and Scorpions. TaqMan probes, for
example are oligonucleotides that have a fl uorescent reporter dye
attached to the 5¢ end and a quencher moiety coupled to the 3¢
end. In the unhybridized state, the proximity of the fl uor and the
quench molecules prevents the detection of fl uorescent signal from
the probe. During PCR, when the polymerase replicates a template
on which a TaqMan probe is bound, the 5¢-nuclease activity of the
polymerase cleaves the probe. This decouples the fl uorescent and
quenching dyes and the fl uorescent dye emits light upon irradia-
tion. Fluorescence increases in each cycle, proportional to the
amount of probe cleavage ( 55 ).
The real-time PCR thermal cycler has a fl uorescence detection
threshold, below which it cannot discriminate the difference
between an amplifi cation generated signal and background noise.
The cycle threshold ( C t ) is defi ned as the number of cycles required
for the fl uorescent signal to cross the threshold (i.e., exceed back-
ground level). C t levels are inversely proportional to the amount of
target nucleic acid in the sample (i.e., the lower the C t level the
greater the amount of target nucleic acid in the sample). Data anal-
ysis, including standard curve generation and copy number calcu-
lation, are performed automatically ( 56 ).

(a) Selection of a suitable internal standard.
An appropriate gene for use as an internal standard and
normalization of data must be selected.
(b) Validation of equal effi ciency of amplifi cation of target gene
and internal standard.
For accurate quantitation, the internal standard must be
amplifi ed simultaneously with the target, be expressed at a con-
stant level and be unaffected by the experimental treatment.
(c) Design of primers and probes to target genes.
Primers and probes may be designed for selected target
sequence. When ordering target gene and internal standard
probes to be used in multiplex reactions, ensure they are labeled
with different reporter dyes.

3.4.1. Steps to Conduct
Before PCR