Biomedical Engineering Reference
In-Depth Information
V
RNA
CQ
=
×
V
Plasma
in which
C
represents the target RNA concentration in plasma (pg/mL for
GAPDH
; copies/mL for
hPL
),
Q
represents the target RNA concentrations of
the extracted RNA samples (pg/
L for
hPL
), which is
determined by a sequence detector in a PCR,
V
RNA
represents the total volume
of RNA obtained after extraction (typically 30
µ
L for
GAPDH
, copies/
µ
L),
V
Plasma
represents the vol-
ume of plasma used for extraction (typically 1.6 mL).
The validations of both the
GAPDH
mRNA
(
11
)
and
hPL
mRNA
(
17
)
real-
time RT-PCR systems are described in previous publications.
µ
4. Notes
1.
Primers and probes are designed with the use of
Primer Express
®
Software v2.0
(Applied Biosystems). Certain precautions for the design are listed as follows:
a. The amplicon length should be less than 100 bp, ideally no longer than 80 bp.
Short amplicon length is preferable for several reasons: (1) the synthetic
oligonucleotide which is used as a calibration curve (
see
Note 3
) is commer-
cially available with size only up to 100 nucleotides. Thus, the amplicon length
is limited to 100 bp; and (2) amplification with shorter amplicon length is
more efficient than that involving longer amplicon length
(
18
)
.
b. Primers for amplification should ideally be intron-spanning in order to mini-
mize any amplification of contaminating genomic DNA. The primer/probe
locations of
GAPDH
and
hPL
mRNA are showed in
Fig. 1
. By using intron-
spanning primers, intron-containing amplification products from the
contaminating genomic DNA are too long to be amplified and thus the ampli-
fication will be cDNA-specific. If the intron/exon junctions are unknown, or
when targeting an intron-less gene, it is necessary to perform DNase treat-
ment in RNA samples (
see
Note 8
).
c. To avoid false-positive results arising from co-amplification of genes with
high homology, it is necessary to perform a BLASTN search with the primer
and probe sequences against the NCBI GenBank. The result of such search
will provide information regarding the specificity of the amplification.
2.
For the
GAPDH
RT-PCR system, the use of synthetic oligonucleotides as the cali-
bration curve (
see
Note 3
) is impossible because of the 226-bp long
GAPDH
amplicon. In this situation, the Human Control RNA is used instead. The manu-
facturer estimates that 1 pg of this control RNA contains approx 100 copies of
GAPDH
transcript.
3.
Generally, in vitro-transcribed RNA is used as a calibration curve for an absolute
RNA quantification. This in vitro-transcribed RNA is usually generated by
subcloning the amplicon behind a T7 RNA polymerase promoter in a plasmid
vector. However, this procedure is labor-intensive and time-consuming, and is
unsuitable when a large number of RT-PCR systems need to be constructed in a
