Biomedical Engineering Reference
In-Depth Information
Results of the singleplex qPCR with EvaGreen
for MLC-2v
Table 5
cDNA
C
T
Efficiency [%]
T
M
[C]
19
VideoScan
16.77
77.2
*83
iQ5
16.54
73
*81
1:1,000
VideoScan
27.12
69.8
*83
iQ5
27.23
69.9
*81
Table 6
Results of a duplex qPCR for the amplification data of genes HPRT1 and MLC-2v
Target
C
T
Efficiency [%]
HPRT1
VideoScan
23.18 ± 1.3
*77
iQ5
25.01 ± 0.7
*88
MLC-2v
VideoScan
27.12 ± 0.9
*75
iQ5
27.03 ± 0.5
*68
(see [
74
-
77
] for details of the chosen targets). Next we performed a qPCR for the
amplification of MLC-2v. The cDNA of heart tissue was used at two concentra-
tions (19, 1:1,000). Despite the different appearance of the amplification curves,
mathematical analysis of the data revealed that the efficiency and C
T
values
(Table
5
) were very similar between both systems independent of the dilution at
given C
T
values (Fig.
18
b). Melting peak analysis confirmed the sole amplification
of MLC-2v.
The duplex TaqMan
qPCR HPRT1 and MLC-2v performed similarly. In both
systems the simultaneous detection of HPRT1 and MLC-2v was possible with
comparable C
T
values (Table
6
). However, it appears that the amplification effi-
ciency was about 10% lower in PCRs with HCU (Table
6
). There was no
amplification detected in non-transcribed samples (not shown).
In conclusion, we have shown that the VideoScan HCU can be used for a broad
range of PCR applications including mPCR, single and duplex qPCR with a
performance comparable to highly specialized technologies. The presence of all
targets in the mPCR indicates that both the iQ5 thermal cycler and the VideoScan
HCU have similar conditions in the reaction vessel. At least for the duplex
TaqMan
qPCR, we observed slightly decreased amplifications efficiencies. This
may in part be explained by minor differences in the temperature offset of 2 Cas
described in
Sect. 6.2
. In its current state the VideoScan system offers only a single
module HCU, which limits the throughput. However, the aim of the HCU is not to
replace existing systems but to offer a platform for other, i.e., microbead-based,
assay formats (see following sections). A multi-vessel version is currently under
development.
