Biochemical and Molecular Biological Assessments of Intracerebral Hemorrhage - Animal Models of Acute Neurological Injuries: Injury and Mechanistic Assessments - page 640

3.6. Real-Time PCR

Real-time PCR is identical to a simple PCR except that the progress
of the reaction is monitored by a camera or detector in “real-time,”
and is regarded as the most sensitive and reproducible form of
PCR-based quantifi cation. There are two methods of real-time
PCR: probe-based and intercalator-based. Both methods require a
special thermocycler equipped with a sensitive camera that moni-
tors the fl uorescence in each well of the 96-well plate at frequent
intervals during the PCR reaction:

Probe-based real-time PCR requires a pair of PCR primers as

●

regular PCR does, an additional fl uorogenic probe which is an
oligonucleotide with both a reporter fl uorescent dye and a
quencher dye attached. Hence, under normal circumstances,
the fl uorescent emission from the probe is low. However, dur-
ing the PCR the probe binds to the gene of interest and becomes
cleaved by the polymerase. Hence, the reporter and quencher
are physically separated and the fl uorescence increases.
Intercalator-based method requires a double-stranded DNA

●

dye (e.g., SYBR green) in the PCR reaction which binds to
newly synthesized double-stranded DNA and gives fl uores-
cence. So as the number of copies of DNA increases during the
reaction so the fl uorescence increases.

3.7. Notes

1. RNases are exceptionally stable, some even resist autoclaving at
120°C for 15 min in super-heated steam. The presence of
ubiquitous and diffi cult to inactivate RNases in the working
environment means that working with RNA is especially prob-
lematic. In effect, contamination of work solutions, work sur-
faces, disposables, consumables, etc. by RNases needs to be
avoided right from the beginning of RT-PCR processing. As a
rough guide to avoiding RNase contamination: use autoclaved
tubes and solutions wherever possible and wear gloves to mini-
mize nuclease contamination from fi ngers (Fig. 2a ).
2. The magnesium concentration is also critical, so care should be
taken that the addition of reagents does not lower the magne-
sium molarity; i.e., some nucleic acid buffers contain 1 mM
EDTA, and this can chelate out much of the magnesium. In
general, try to keep the free magnesium concentration at about
2 mM ( 10 ).
3. Choose your PCR primers to be about 18-22 bases in length
and not too high or low in G + C content.

3.8. Protocol for
Real-Time PCR

This protocol describes the detailed procedure for RT-PCR using
SYBR Green I for quantitative analysis of hemoglobin gene expres-
sion ( 11 ). The procedure begins with reverse transcription of total
RNA. The cDNA is then used as the template for real-time PCR
with gene-specifi c primers.