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Real Time PCR (RT-PCR)
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- The real time PCR allows the relative or absolute quantification of messenger RNA (mRNA) from a given simple. The starting material can be both total or messenger RNA. In both cases, the RNA is retrotranscribed into cDNA.
- The methodology consists in designing specific primers to amplify by PCR the fragment of interest. The fragment’s expression is detected through the fluorescence emitted by the interspersed molecule in the cDNA (such as SYBRGREEN) or using specifics probes (such as Taqman’s probes). After a PCR’s cycle the emitted fluorescence is measured and a curve is obtained when all cycle are finnished (usually around 40). This curve is characterised by a value that is called Ct (cycle threshold), whose low values correspond to high expression.
- Normally it is detected the expression level of one fragment only. However, simultaneous fragment from a simple can be detected, but a specific fluorochrome for each fragment is required.
- The absolute quantification requires the computation of a standard curve based on different concentrations from the fragment to be amplified.
- Apart from gene expression assays, SNPs detection and DNA methylation analysis can be also carried out .
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EQUIPMENT
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CFX96TM i C1000TM (BioRad)
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ASSAY SUPPORT
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1 plate de 96 wells
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SAMPLES
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The number of samples per day depends on the PCR program to be used
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CAPACITY
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96 samples simultaneously
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RESULTS
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Files sent by e-mail and/or use of SGB’s software
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ACTIVITY
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CONDITIONS
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Expression assay
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Two assays are possible: one or two steps. The first one consists in retrotranscribe the RNA and PCR without interruption. If the assay is two steps, these two steps are separately performed. In that case, the user can run the retrotranscription at its laboratory
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Mutation’s detection
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There are specific assays to detect single nucleotide polymorphisms (SNPs) and DNA methylation. This mutation is based on the different melting temperatura displayed by products that are generated during a PCR. This technique is called High Resolution Melting (HRM).
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PCR
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Each user can customise the design of its the PCR program.
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Analysis
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CFX Manager software .
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