Biomedical Engineering Reference
In-Depth Information
the change in fl uorescence as a function of temperature from a dye or probe
interacting with the double-stranded DNA. This is especially relevant if
SYBR Green is the dye being used for product detection, as the real-time
profi les do not distinguish between specifi c amplicon and nonspecifi c products
(e.g., primer-dimers). The dissociation profi le can also be useful in primer
concentration optimization.
4. Notes
1. Reagents listed in
Subheading 2
are by no means the only reagents that can be
used. There are multiple vendors for RNA purifi cation reagents, RT and PCR
enzymes and reagents, and SYBRGreen master mix solutions. The authors of this
chapter make no specifi c endorsement of any one reagent. We present these only
as a point of reference for how the experiments herein were performed.
2. There are multiple protocols available to do quantitative PCR (
13
,
14
; a simple
Medline search for
real-time PCR
and
quantitation
gave >800 citations), and
which one you chose will depend on your specifi c application.
3. RNA is notoriously unstable and prone to degradation, so the utmost care must be
taken when working with it. Any RNA isolation reagent will likely be provided
with detailed instructions for working with RNA.
4. The effi ciency of the reverse transcription reaction is by no means uniform across
all samples and using all methods. In addition, depending on the report, people
use oligo-dT, random hexamers, or gene-specifi c primers to produce the cDNA.
Each choice comes with advantages and disadvantages. We fi nd that if RNA
is not limiting (increasing signal-to-noise ratios and decreasing nonspecifi c
signals), then gene-specifi c reverse priming is the most desirable.
5. ABI recommends primers that produce amplicons of ~100 basepairs (bp)
for maximum effi ciency of the PCR reaction; however, we routinely use prim-
ers designed by Clontech (amplicons are 200-400 bp) without any apparent
problems.
6. RT-PCR can be performed as a one-step or two-step reaction. We prefer two-step
RT-PCR, largely because it makes maximal use of precious RNA samples (a
single cDNA can be used in multiple subsequent PCR reactions). In addition,
a relative standard curve can be easily derived from a cDNA preparation by
performing a dilution series. Finally, two-step reactions are superior in detecting
low abundance transcripts and therefore best to use when doing new assays with
unknown abundance transcripts.
Fig. 2.
(previous page)
Real-time PCR analysis of rat brain RNA levels (of the
nerve growth factor [NGF] gene). Plot of a dilution series (1, 1
8) of cDNA
derived in the fi rst-strand synthesis reaction. Each dilution was assayed in triplicate.
The line at
2, 1
4, 1
Rn = 1 (center) represents the
C
t
for each reaction. Average
C
t
values:
1
1
C
t
= 24.1 ± 0.169; 1
2
C
t
= 25.1 ± 0.024; 1
4
C
t
= 26.2 ± 0.109; and 1
8
C
t
= 27.3 ± 0.143.
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