|
Gene
Expression Assays (mRNA)
Transfection Efficiency Assays
Nuclear Run-on Assays
Gene
Dosage Assays
Contact
Gene
Expression Assays (mRNA)
The Functional Genomics Core Laboratory uses an ABI PRISM 7700
Sequence Detection System, fluorogenic TaqMan‰ probes, and
specific PCR primers to quantitate specific mRNA levels. The Perkin-Elmer/Applied
Biosystems (PE/ABI) Prism 7700 Sequence Detection System allows
relative or absolute quantitation of mRNA levels. This instrument
monitors PCR at every cycle using a closed-tube fluorogenic 5’
nuclease-based assay, and utilizes a fluorogenic probe consisting
of an oligonucleotide with both a reporter and a quencher dye
attached. During PCR, the probe anneals to the target of interest
specifically between the forward and reverse primers. If hybridization
occurs, the probe is cleaved by the 5’ nuclease activity
of the Taq DNA polymerase. This separates the reporter from the
quencher dye and a sequence-specific fluorescence signal is generated.
The signal is proportional to the amount of PCR product formed
and is measured continuously throughout the entire PCR assay (real
time). This technique further increases the specificity of the
PCR reaction beyond the level achieved with gene specific PCR
primers. A mismatch between probe and target substantially reduces
the efficiency of hybridization and cleavage; therefore, a mismatched
probe does not contribute appreciably to the measured fluorescent
signal. In addition, since fluorescence signal is generated only
if the target sequence for the probe is amplified during PCR,
no signal is generated by non-specific amplification. This direct
detection together with the 96-well microtiter plate format enables
a cost-efficient and extremely high-throughput platform.
Since
2000, the Functional Genomics Laboratory has developed over 115
TaqMan-based, quantitative, gene-expression assays and analyzed
over 17,000 samples.
Transfection
Efficiency Assays
The Functional Genomics Core Laboratory uses an
ABI PRISM 7700 Sequence Detection System, fluorogenic TaqMan™probes,
and specific PCR primers to quantitate transfection efficiency.
Nuclear
Run-on Assays
The Functional Genomics Core Laboratory uses an
ABI PRISM 7700 Sequence Detection System, fluorogenic TaqMan™probes,
and specific PCR primers to quantitate mRNA processing.
Gene
Dosage Assays
The Functional Genomics Core Laboratory uses an
ABI PRISM 7700 Sequence Detection System, fluorogenic TaqMan™probes,
and specific PCR primers to characterize haploinsufficiency or
gene dosage. This assay is helpful in improving the efficiency
of evaluating and developing transgenic animals.
|
