Biochemical and Molecular Biological Assessments of Intracerebral Hemorrhage - Animal Models of Acute Neurological Injuries: Injury and Mechanistic Assessments - page 639

Most of these use some form of affi nity purifi cation, where disruption
of the cell or nucleus is followed by nucleic acid binding to a solid
support.

3.2. Reverse
Transcription
and cDNA Synthesis

In order to be successfully amplifi ed by PCR, RNA needs to be fi rst
converted into a DNA copy (cDNA), a process most effi ciently
achieved using an RNA-dependent DNA polymerase enzyme
(reverse transcriptase, RT).
The most popular method for reverse transcription of RNA
into cDNA, initially involves making two different reverse transcrip-
tion reaction mixes ( 8 ). The fi rst mix includes the isolated RNA
template and the primer to be used to initiate reverse transcription,
while the second mix includes dNTPs (dATP, dCTP, dGTP, and
dTTP), RNase inhibitor, RT enzyme, RT buffer (including the
appropriate concentration of magnesium ions), dithiothreitol, and
nuclease-free buffer. The fi rst mix (containing RNA and RT primer)
is incubated at 80°C for 2 min and then immediately placed on ice,
which helps remove and “freeze open” secondary structure within
the RNA, increasing the specifi city of primer binding. After 5 min
on ice, the second RT mix is added to the fi rst mix and the new
complete RT mix is incubated at 37°C for 1 h. After 1 h, the resul-
tant reverse transcription products may be directly added to the
appropriate PCR mix and PCR cycling performed.

A typical PCR reaction mix includes target DNA, specifi cally designed
oligonucleotide primers, deoxyribonucleotide triphosphates/
magnesium ions/a buffer component, a thermostable DNA poly-
merase, and water.

3.3. The PCR Reaction
Mix

3.4. Cycling
Parameters

In a typical reaction, the double-stranded DNA is denatured by
briefl y heating the sample to 90-95°C. The primers are allowed to
anneal to their complementary sequences by briefl y cooling to
40-60°C, followed by heating to 70-75°C to extend the annealed
primers with the Taq polymerase ( 9 ).
For many PCR applications, 20-30 cycles of denaturation,
annealing, and chain extension are suffi cient to generate visible
amplifi cation products using gel electrophoresis in combination
with ethidium bromide staining. To reduce the likelihood of non-
specifi c hybridization between primers and regions of DNA which
contain similar but not identical DNA sequences, both annealing
and elongation steps of each thermocycle should be performed at
“stringent” temperatures.

3.5. Analysis of PCR
Amplifi cation Products

The visualization of PCR amplifi cation products is traditionally
performed by gel electrophoresis and ethidium bromide staining, a
chemical which inserts between the two DNA strands and is detected
by fl uorescence during exposure to ultraviolet (UV) light.