Biomedical Engineering Reference
In-Depth Information
Fig. 1.
(A)
Locations of primers and probe for
glyceraldehyde-3-phosphate dehy-
drogenase
(
GAPDH
) mRNA.
(B)
Locations primers, probe and synthetic DNA oligo-
nucleotide for
human placental lactogen
(
hPL
) mRNA.
short period of time. An alternative method that is increasingly used is to con-
struct a calibration curve with the use of synthetic single-stranded oligonucle-
otides specifying the various amplicons. Previous data have shown that such
single stranded oligonucleotides reliably mimic the products of the reverse tran-
scription step and produce calibration curves that are identical to those obtained
using T7-transcribed RNA
(
18
)
. The synthetic oligonucleotide for hPL mRNA is
4.
Our studies have revealed that 1.6 mL of plasma is the minimal sample volume for
robust plasma RNA detection for many RNA targets. Thus, we strongly suggest
that at least 4 mL of blood samples should be collected. For the
GAPDH
RT-PCR
system, the minimal plasma sample volume is 0.6 mL.
5.
When the blood samples are left unprocessed, their corresponding plasma and
serum RNA concentrations will fluctuate over time
(
12
)
. This artifactual fluctua-
tion may be due to several factors, such as release of RNA from necrotic and/or
apoptotic blood cells, and the stability of the original and the newly released RNA.
To guarantee a reliable plasma and serum RNA concentrations, we recommend all
blood samples, including EDTA blood and clotted blood, be stored at 4°C and be
processed within 6 h.
6.
Based on our previous report that blood
processing by different protocols would
affect the result of plasma DNA quantitation
(
20
,
21
)
, our current protocol
involves an initial 1600
g
centrifugation of blood samples followed by a second
centrifugation at 16,000
g
. This protocol has been shown to minimize the contribu-
tion
of RNA and DNA derived from residual cells in plasma.
7.
A recent study has shown that RNA in frozen plain plasma would degrade with
time
(
22
)
. Therefore, to preserve the integrity of RNA, Trizol LS reagent should
be added to the plasma before storage if the sample is to be archived
(
22
)
. Based
on our studies (unpublished), if RNA extraction would be performed within 2 wk, it
is acceptable to store plasma at the following conditions: (1) -80°C with or with-
