Measurement of MDRI Gene Expression by Real-Time Quantitative RT-PCR Using the LightCycier instrument m
CyS-labeled primer and the fluorescein-labeled probe were designed to allow
fluorescence resonance energy transfer (FRET) to occur when the hybridization
probe annealed to the PCR products. The FRET was monitored by LightCycler
in a real-time fashion (see "Results" for detail) for quantification purposes
(Table 1).
Table 1. Oligonucleotides
MDRI GcnBanl< Accession #M29428
Primers
CAGGAGATAGGCTGGTTTGAXGTa
TTAGCTTCCAACCACGTGTAAATC
Hybridization probe
GTCGGGTGTTAAGCTCCCCAACATCG
(3' -fluorescein-labeled)
Product
• X. modified T with Cy5 attached.
Hybridization Probe Master Mix:
dNTPs (2 mM stock)
MgCl2 (40 mM)
Forward primer (5 fJM each)
Reverse primer (211M)
Hybridization probe (1 fLM)
Diluted enzyme + TaqStart anibod}'"
Sterile Glass Distilled H2O
Total volume
Region
151 - 173 (Exon 6)
840- 863 (Exon 7)
177-202
Length GC (%)
Tm (0C)
23
24
26
172
48
42
58
64.6
63.8
71.6
Volume [JIl]
[Final]
1.0
200 fJM
1.0
4mM
1.0
0.5 fJM
1.0
0.2 fJM
1.0
0.1 fJM
1.0
0.4 U Taq and 8.8 ng
TaqStart antibody
3.0
9
• Diluted enzyme wa. s prepared by adding 1 fLl of Promega Taq (5 UIIlI) to 1 fLl of ClonTech
TaqStart antibody (J 10 ng/Ill). This mixture was incubated at room temperature for 5 min
prior to the addition of 10.5 fLl enzyme diluent. This solution was then added to the master mix.
In total, 9 fJl of master mix and 1 fJl of samples were added to the capillary tubes
which were sealed, centrifuged in a micro centrifuge, and placed in the LightCycler rotor.
LightCycier PCR
CyS-labeled primer and the fluorescein-labeled probe were designed to allow
fluorescence resonance energy transfer (FRET) to occur when the hybridization
probe annealed to the PCR products. The FRET was monitored by LightCycler
in a real-time fashion (see "Results" for detail) for quantification purposes
(Table 1).
Table 1. Oligonucleotides
MDRI GcnBanl< Accession #M29428
Primers
CAGGAGATAGGCTGGTTTGAXGTa
TTAGCTTCCAACCACGTGTAAATC
Hybridization probe
GTCGGGTGTTAAGCTCCCCAACATCG
(3' -fluorescein-labeled)
Product
• X. modified T with Cy5 attached.
Hybridization Probe Master Mix:
dNTPs (2 mM stock)
MgCl2 (40 mM)
Forward primer (5 fJM each)
Reverse primer (211M)
Hybridization probe (1 fLM)
Diluted enzyme + TaqStart anibod}'"
Sterile Glass Distilled H2O
Total volume
Region
151 - 173 (Exon 6)
840- 863 (Exon 7)
177-202
Length GC (%)
Tm (0C)
23
24
26
172
48
42
58
64.6
63.8
71.6
Volume [JIl]
[Final]
1.0
200 fJM
1.0
4mM
1.0
0.5 fJM
1.0
0.2 fJM
1.0
0.1 fJM
1.0
0.4 U Taq and 8.8 ng
TaqStart antibody
3.0
9
• Diluted enzyme wa. s prepared by adding 1 fLl of Promega Taq (5 UIIlI) to 1 fLl of ClonTech
TaqStart antibody (J 10 ng/Ill). This mixture was incubated at room temperature for 5 min
prior to the addition of 10.5 fLl enzyme diluent. This solution was then added to the master mix.
In total, 9 fJl of master mix and 1 fJl of samples were added to the capillary tubes
which were sealed, centrifuged in a micro centrifuge, and placed in the LightCycler rotor.
LightCycier PCR