Biomedical Engineering Reference
In-Depth Information
elevated in the plasma of cancer patients
(
11
)
indicates that caution should be
taken when expressing or interpreting plasma RNA data as a ratio. As quantifi-
cation of mRNA transcript can be either relative or absolute, to avoid the inap-
propriate normalization in relative quantification, Lo and his colleagues have
further developed a real-time quantitative RT-PCR for absolute quantification
of circulating RNA in plasma. The latter group has further demonstrated that
placental mRNA transcripts in maternal plasma are readily detectable, and the
placenta is an important source of fetal RNA in maternal plasma
(
17
)
. This
study represents the first quantitative analysis of gene expression of the fetus
by analyzing a plasma sample from the mother. In this first report, the absolute
calibration curve was constructed by serial dilutions of high-performance liquid
chromatography (HPLC)-purified single-stranded synthetic DNA oligonucle-
otides specifying the studied amplicon. Previous data have shown that such
single stranded oligonucleotides reliably mimic the products of the reverse
transcription step and produce calibration curves that are identical to those
obtained using T7-transcribed RNA
(
18
)
. The use of such calibration method-
ology poses certain advantages: first, it provides absolute concentration of
mRNA transcript in plasma regardless the utility of the housekeeping gene
normalization; and second, as a result of the commercial availability of the
synthetic oligonucleotides, it significantly simplifies the process of obtaining a
calibration curve when compared with the labor-intensive preparation of cali-
bration curve involving amplicon subcloning and in vitro transcription.
The two systems discussed as follows are based on our recent development
of the absolute quantifications of circulating RNA in plasma. The
GAPDH
RT-PCR system aims to quantify the housekeeping gene transcript,
GAPDH
,
present in the plasma of human subjects
(
11
,
12
,
17
)
. This can be used as a posi-
tive control system to verify the quality of extracted RNA from plasma. The
second RT-PCR system aims to measure the concentration of placenta-
expressed gene transcript,
human placental lactogen
(
hPL
), present in the
plasma of pregnant women
(
17
)
. This
hPL
RT-PCR system can be potentially
used in noninvasive prenatal monitoring.
2. Materials
2.1. Sample Collection
Ethylenediamine tetraacetic acid (EDTA)-containing blood collection tubes
for plasma collection.
2.2. RNA Extraction
1.
Trizol LS reagent (Invitrogen, Carlsbad, CA).
2.
Chloroform.
3.
Ethanol.
4.
RNeasy Mini Kit (Qiagen, Hilden, Germany).
