Quantitative Two·Step RT· PCR for the Detection of Human ABCA 1 Transporter on LightCyder n
quality, non-degraded RNA. Concentration is calculated from the fluorescence
signal obtained. Only high-quality RNA was used.
ABCAI-specific PCR primers and hybridization probes, capable of fluorescence
resonance energy transfer (FRET) were used to generate and monitor a 205-bp
ABCAI fragment and a 282-bp porphobilinogen deaminase fragment (PBGD),
respectively (Table O. Primers and hybridization probes for ABCAI and PBGD
were synthesized by TIB MOLBIOL. Hybridization probes were labeled with fluorescein at the 3' end of the donor probe and LCRed640 at the 5' end of the acceptor probe.
A 205-bp ABCAI RT-PCR product was sub cloned in the plasmid vector pCRR 11TOPO containing a T7 RNA polymerase promoter (TOPO TA Cloning, Invitrogen) according to the manufacturer's instructions. Isolation and purification of
plasmid DNA was performed using the Qiaprep Spin Miniprep Kit (QIAGEN,
Germany). The ABCAI insert sequence was verified by DNA sequencing using an
ABI Prism Genetic Analyzer 310 (PE Biosystem). Linearization of purified plasmid DNA was achieved by incubation with BamHI and RNA was synthesized in
vitro using the RiboProbe R In Vitro Transcription System (Promega) and T7 RNA
Polymerase following the protocol for large scale RNA synthesis.
First-strand cDNA synthesis was performed in a total volume of 20 III using 40 U
AMV Reverse Transcriptase, 2.0 III lOx Reaction Buffer, 50 U RNase Inhibitor, 2 III
Deoxynucleotide Mix (1 mM),2 j.1l Random Primer p(dN)6 (3.2 Ilg), 4 III MgCl z
Table 1. Oligonucleotides
GcnBank Accession #AB055982 (human ABCAI mRNA),X04217 (human PSGO mRNA)
Position
Length GC (%)
Tm (0C)
ABeAl forward primer
GCACTGAGGAAGATGCTGAAA
1013
21
47.6
60,3
ABeAl reverse primer
AGTTCCTGGAAGGTCTTGTTCAC
1217R
23
47.8
60,8
ABeAl donor probe
GCCGCTGCTCGTTGGGAAGAT -F
1128
21
61.9
70,1
ABeAI acceptor probe
LCRed640-CTGTATACACCTGACACTCCAG-P
1150
22
50.0
54,4
PBGD forward primer:
AGAGTGATTCGCGTGGGTACC
85
21
57.1
63,5
PBGD reverse primer:
GGCTCCGATGGTGAAGCC
366
18
66.7
64,0
PBGD donor probe:
AGTGGACCTGGTIGTTGCACTCCTIGAA-F
297
28
48.1
72,3
PBGD acceptor probe:
LCRed640-ACCTGCCCACTGTGCTTCCTCCT -P
326
23
60.9
69,7
Oligonucleotide
Design
Generation of an
External ABCAl RNA
Standard
Reverse
Transcription
quality, non-degraded RNA. Concentration is calculated from the fluorescence
signal obtained. Only high-quality RNA was used.
ABCAI-specific PCR primers and hybridization probes, capable of fluorescence
resonance energy transfer (FRET) were used to generate and monitor a 205-bp
ABCAI fragment and a 282-bp porphobilinogen deaminase fragment (PBGD),
respectively (Table O. Primers and hybridization probes for ABCAI and PBGD
were synthesized by TIB MOLBIOL. Hybridization probes were labeled with fluorescein at the 3' end of the donor probe and LCRed640 at the 5' end of the acceptor probe.
A 205-bp ABCAI RT-PCR product was sub cloned in the plasmid vector pCRR 11TOPO containing a T7 RNA polymerase promoter (TOPO TA Cloning, Invitrogen) according to the manufacturer's instructions. Isolation and purification of
plasmid DNA was performed using the Qiaprep Spin Miniprep Kit (QIAGEN,
Germany). The ABCAI insert sequence was verified by DNA sequencing using an
ABI Prism Genetic Analyzer 310 (PE Biosystem). Linearization of purified plasmid DNA was achieved by incubation with BamHI and RNA was synthesized in
vitro using the RiboProbe R In Vitro Transcription System (Promega) and T7 RNA
Polymerase following the protocol for large scale RNA synthesis.
First-strand cDNA synthesis was performed in a total volume of 20 III using 40 U
AMV Reverse Transcriptase, 2.0 III lOx Reaction Buffer, 50 U RNase Inhibitor, 2 III
Deoxynucleotide Mix (1 mM),2 j.1l Random Primer p(dN)6 (3.2 Ilg), 4 III MgCl z
Table 1. Oligonucleotides
GcnBank Accession #AB055982 (human ABCAI mRNA),X04217 (human PSGO mRNA)
Position
Length GC (%)
Tm (0C)
ABeAl forward primer
GCACTGAGGAAGATGCTGAAA
1013
21
47.6
60,3
ABeAl reverse primer
AGTTCCTGGAAGGTCTTGTTCAC
1217R
23
47.8
60,8
ABeAl donor probe
GCCGCTGCTCGTTGGGAAGAT -F
1128
21
61.9
70,1
ABeAI acceptor probe
LCRed640-CTGTATACACCTGACACTCCAG-P
1150
22
50.0
54,4
PBGD forward primer:
AGAGTGATTCGCGTGGGTACC
85
21
57.1
63,5
PBGD reverse primer:
GGCTCCGATGGTGAAGCC
366
18
66.7
64,0
PBGD donor probe:
AGTGGACCTGGTIGTTGCACTCCTIGAA-F
297
28
48.1
72,3
PBGD acceptor probe:
LCRed640-ACCTGCCCACTGTGCTTCCTCCT -P
326
23
60.9
69,7
Oligonucleotide
Design
Generation of an
External ABCAl RNA
Standard
Reverse
Transcription