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Real-Time PCR
REAL-TIME PCR
Real-time reverse transcription (RT) polymerase
chain reaction (PCR) is the most sensitive and reliable method for the detection
and quantitation of nucleic acids levels. The Affymetrix Resource Facility has
acquired the ABI 7900 sequence detection system which allows real time detection
of PCR products as they accumulate during PCR and thus enables the accurate
quantitation of DNA and RNA over a wide dynamic range. The ABI 7900 instrument
can be used for various applications such as quantitation of gene expression,
allelic discrimination (SNP detection) and pathogen detection.
The
DDCt
method of RT-PCR is ideal for primer pairs with an efficiency of 90% or greater
AND for genes that will show a large fold change in gene expression (5-fold or
greater). The standard curve method will control for differences in primer
efficiency. If the fold change data from a standard curve run is in
agreement with DDCt-calculated
fold changes, then subsequent plates of RT-PCR using the same primer pairs can
be run using the DDCt
method.
Quantitative Real-Time PCR
Allelic Discrimination Assay (SNP
Genotyping)
Primer
and Probe Design
Submission
Service Fees
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