Biomedical Engineering Reference
In-Depth Information
existence of circulating RNA in plasma was first reported in 1999 when Lo et
al. observed the presence of the tumor-associated RNA in the plasma of patients
with nasopharyngeal carcinoma
(
1
)
. In conjunction with the report by Kopreski
et al.
(
2
)
, these publications represent the first demonstrations of reverse-tran-
scription (RT)-polymerase chain reaction (PCR)-amplifiable tumor-derived
RNA in the plasma of cancer patients. Since then, tumor-associated RNA
targets that have been detected include mRNA of the telomerase components,
mammaglobin
,
-catenin
,
CK19
,
huRNP-B1
,
Her2/neu
, and
CEA
(
3-8
)
. In ad-
dition, the availability of such an approach has stimulated the first dem-
onstration of the presence of fetal RNA in maternal plasma
(
9
)
.
Initially, the lability of RNA and the existence of ribonuclease in the plasma
(
10
)
make it surprising that circulating RNA should be detectable at all. How-
ever, recent demonstration by Ng and his colleagues showed that a significant
portion of circulating RNA in plasma is associated with subcellular particles
(
11
)
. This particle-associated nature of circulating RNA may explain the sur-
prising stability of such RNA in plasma
(
12
)
. As a result of this inherent stabil-
ity of circulating RNA, the storage of plasma samples at -80°C will be
sufficient to reduce degradation, and the development of robust RNA extrac-
tion from plasma becomes realistic.
For most of the published works on the detection of circulating RNA in
plasma, techniques such as conventional RT-PCR that are relatively sensitive
and specific have been used. However, this approach is limited to qualitative
analysis and requires
time-consuming post-PCR analysis, making the routine
implementation of such methodology difficult. Real-time RT-PCR, on the other
hand, which is a quantitative approach, is increasingly used for measuring the
level of gene expression. This technique is based on the performance of
RT-PCR in the presence of a dual-labeled fluorescent probe, which allows the
fluorescence signals
to be recorded and analyzed during PCR cycling
(
13
)
.
Together with suitable instrumentation, the steps of amplification, detection,
and quantification can be combined. Thus, this methodology runs as a closed-
tube system and postamplification manipulation can be eliminated. Further-
more, it eliminates risk of contamination and minimizes hands-on time. When
used appropriately, this technique is robust and highly suited for high-through-
put screening application
(
14
,
15
)
.
With the use of the real-time quantitative RT-PCR, Dasi et al. have dem-
onstrated that the concentration of
telomerase reverse transcriptase
mRNA in
plasma was elevated in colorectal cancer patients
(
16
)
. This study employed
relative quantification of mRNA, in which the results were expressed as a ratio
of
telomerase
mRNA to
glyceraldehyde-3-phosphate
dehydrogenase
(
GAPDH)
mRNA so as to normalize plasma RNA levels from different individuals. How-
ever, recent demonstration by Lo's group that the concentrations of particle-
associated and nonparticle-associated
GAPDH
mRNA were significantly
β
