Biomedical Engineering Reference
In-Depth Information
Table 4
Primers for the amplification of PCR products from human heart tissue
Gene
Primer
GAPDH
§
f: TGCACCACCAACTGCTTAGC
¥
, r: GGCATGGACTGTGGTCATGAG
¥
HPRT1
§
f: GGCAGTATAATCCAAAGATGGTC, r: TTCAAATCCAACAAAGTCTGGC
d: Atto465-AGGTCGCAAGCTTGCTGGTGAAAAGG-BHQ1
VIM
f: CCCTTGACATTGAGATTGCC, r: CCAGATTAGTTTCCCTCAGGT
cTnT
#
f: AGTGGGAAGAGGCAGACTGA
#
, r: CGAACTTCTCTGCCTCCAAG
#
SERCA2
#
f: GTCACTCCACTTCCTGATCC
#
, r: CTCCAGTATTGCAGGTTCCA
#
MLC-2v
#
f: ACAGGGATGGCTTCATTGAC
#
, r: ATGCGTTGAGAATGGTTTCC
#
d: Atto647N-CAGGGTCCGCTCCCTTAAGTTTCTCC-BHQ2
ADRB1
*
f: AGGGATTTCTACCTCACACTG, r: ACAGAGTCACATGTCACAGAG
ADRB2
*
f: GATTTCAGGATTGCCTTCCA, r: GTACTACAATTCCTCCCTTGTG
ET
*
f: AATTGTTTCCAGTCATGCCTC, r: AGTTCATGCTGTCCTTATGG
ET
*
f: TCTGTTGGTATTGGACTATATTGG, r: AGCATAAGCATGACTTAAAGCAG
Forward primer (f), reverse primer (r), TaqMan
hydrolysis probe (d) # [
72
], ¥ [
73
]. Primers for
GAPDH, HPRT1 and VIM were used at 100 nM, hydrolysis probes at 125 nM and the remaining
primers at 400 nM final concentration. *: human receptor, #: cardiac specific gene, §: reference
gene
Fig. 18 Comparison of the PCR performance between the iQ5 thermal cycler (iQ5) and the
VideoScan HCU. a Representative agarose gel: mPCR for human (upper part ) GAPDH, VIM,
cTnT, SERCA2, MLC-2v and (lower part) GAPDH, ET
B
, ADRB1, ET
A
, ADRB2 was amplified
with the iQ5 and VideoScan HCU. All targets were amplified similarly, indicating that the
VideoScan HCU is comparable to the iQ5. b Amplification plot of qPCR with EvaGreen
: The
amplification of MLC-2v. Dashed lines ''iQ5'' thermal cycler, solid lines VideoScan (VS HCU).
Two cDNA concentrations (19, 1:1,000) were used. The inset shows two melting peaks in
reverse-transcribed samples. The non-reverse-transcribed samples remained negative
iQ5 thermal cycler (Fig.
18
a). There was no amplification detected in non-reverse-
transcribed samples. Thus the VideoScan HCU can be used for qualitative
multiplex gene expression analysis of human receptors and cardiac-specific genes
