Biomedical Engineering Reference
In-Depth Information
6.1 PCR in Solution
Quantitative real-time PCR (qPCR) is a well established technology for the
simultaneous amplification and quantification of PCR product synthesis in a
homogeneous format [
63
]. The scope of this technology includes gene expression
analysis, pathogen detection and SNP analysis. Non-specific qPCR chemistries
make use of DNA intercalators like EvaGreen
(Biotium) which reversibly bind to
DNA without sequence selectivity [
64
]. This cheap and simple chemistry is
optimal for single plex qPCRs. For multiplex qPCRs, specific chemistries which
make use of probes are required. Hydrolysis (TaqMan
) probes play a dominant
role and are regarded as one of the simplest and best chemistries available [
65
].
The introduction of multiplex PCRs has led to cost savings (hands-on time,
reagents), savings of precious sample material and increased reliability since
endogenous controls are included [
63
,
66
-
69
].
We characterized the VideoScan HCU in solution by performing mPCRs,
singleplex qPCRs and duplex qPCRs. In proof-of-principle experiments we used a
set of human receptors and cardiac-specific genes as target. The VideoScan HCU
performance regarding C
T
values, amplification efficiency and feasibility of
melting curve analysis was compared to the iQ5 (Bio-Rad Laboratories) real-time
thermal cycler. We chose the iQ5 thermal cycler based on design similarities, i.e.,
the Peltier-based technology for temperature regulation [
67
].
The human heart sample was obtained from the explanted heart of a patient
suffering from dilated cardiomyopathy [
70
] and transplanted in ''Deutsches
Herzzentrum Berlin'' after written informed consent. Total RNA was isolated
using peqGOLD TriFast
TM
(PEQLAB Biotechnologie GmbH) as described by the
manufacturer. The cDNA was synthesized after DNase I (Invitrogen) digestion
from 1 lg total RNA by using the SuperScript II synthesis kit (Invitrogen) with
oligo(dT)
18
primer according to the manufacturer's instructions.
The iQ5 thermal cycler (Eppendorf) and the VideoScan HCU were run in
parallel. PCR reactions were conducted in 20 lL Maxima PCR buffer (Fermentas)
as follows: initial denaturation at 95 C for 10 min, followed 50 PCR cycles of 40 s
at 95 C, 90 s at 58.5 C, and 90 s at 68.5 C. During mPCR (I)GAPDH, VIM,
cTnT, SERCA2 and MLC-2v or (II)GAPDH, ADRB
1/2
, ET
A/B
(Table
4
) were
amplified. In qPCR experiments, fluorescence was measured during the annealing
stage. EvaGreen
(Biotium) was used to monitor the amplification of MLC-2v
from diluted cDNA in a singleplex qPCR. For duplex TaqMan
qPCR, HPRT1
and MLC-2v were used as target.
Raw fluorescence VideoScan data were processed by background fluorescence
subtraction utilizing a custom-made plugin for RKWard [
27
]. The amplification
efficiencies and C
T
values were determined using the dedicated functions from the
qpcR package [
71
]. Amplicons were separated in 3% TBE agarose gel.
The multiplex amplification of (I)GAPDH, VIM, cTnT, SERCA2, MLC-2v or
(II)GAPDH, ADRB
1/2
, ET
A/B
from heart tissue cDNA performed with the Video-
Scan HCU showed an identical pattern compared to results obtained with the
