Biomedical Engineering Reference
In-Depth Information
out Trizol LS reagent, or (2) -20°C only with Trizol LS reagent. If the plasma
samples are stored without Trizol LS reagent, it should be added immediately
before the addition of chloroform during RNA extraction. However, we observed
significant drops in RNA concentration when plasma was stored at -20°C without
Trizol LS reagent even when stored even for 2 wk.
8.
Any significant DNA contamination in RNA samples will result in inaccurate
RNA quantification particularly when non-intron-spanning primers are used.
DNase I treatment must be carried out to eliminate any residual genomic DNA left
during RNA extraction. In addition, it is necessary to test the RNA samples to
ensure they are negative for DNA by substituting the r
Tth
polymerase with the
Ampli
Taq
Gold enzyme (Applied Biosystems). For buffy coat or whole blood
samples, the “On-Column DNase Digestion” appears not to completely remove
all genomic DNA. Other stronger DNase digestion method, such as deoxyribonu-
clease I from Invitrogen, can be used instead.
9.
The use of one-step one-enzyme RT-PCR with r
Tth
polymerase has several
advantages over the two-enzyme RT-PCR:
a. It has been reported that
Tth
polymerase is more resistant to inhibitors present
in biological specimens than
Taq
polymerase
(
23
)
.
b. The thermostable nature of r
Tth
polymerase allows the RT to be performed at
a high temperature of 60°C. This minimizes secondary structures present in
RNA as well as reduces primer dimers and nonspecific bindings during the RT
reaction.
c. In two-enzyme RT-PCR, the PCR product carryover prevention enzyme,
uracil-
N
-glycosylase (UNG), cannot be used because the substitution of dUTP
for dTTP in the RT reaction makes the nascent cDNA a substrate for UNG. On
the other hand, the thermostable r
Tth
polymerase permits the use of UNG
becasue the RT temperature of 60°C inactivates the UNG activity. As the RT
reaction and PCR are carried out together in a single tube, this reduces both the
hands-on time and the risk of contamination.
Acknowledgments
This work was supported by a Earmarked Research Grant (CUHK 4474/
03M) from the Research Grants Council of the Hong Kong Special Adminis-
trative Region (China).
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