Current Applications in Quantitative RealTime PCR

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Current Applications in Quantitative Real-Time PCR

• Craig Osborne
• Nevada Genomics Center
• genomics_at_unr.nevada.edu

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Starting Materials

• Structural Assay
• Uses DNA, typically genomic extractions
• Single Nucleotide Polymorphism
• Functional Assay
• Uses RNA extractions
• Uses reverse transcriptase to generate cDNA
  templates
• Differential expression
• Diagnostics involving gene expression
• RNA interference

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RT-PCR Chemistries

• 5 exonuclease
• TaqMan
• Requires primers and labeled probe specific to
  gene
• Intercalation
• SYBR Green
• Requires primers specific gene
• Labeled Primer
• Light Upon Extension (LUX)
• Requires labeled primer that contains a hairpin
  loop

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5 Exonuclease
5
5 Exonuclease

• Advantage
• Probe is specific to template
• Disadvantage
• Cost
• Probe degradation

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Intercalation

• SYBR Green
• Binds to minor groove of double stranded DNA
• Can only be fluoresced when bound
• Advantage
• Low cost
• Disadvantage
• Binds to all dsDNA in the reaction

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Quantitation Strategies

• Absolute quantitation Unknown samples are
  compared to a standard curve. The standard is a
  known DNA sample whose concentration is known
  absolutely. The accuracy of the absolute
  quantitation assay is entirely dependent on the
  accuracy of the standards.
• Relative quantitation A comparison within a
  sample (DNA or cDNA) is made with the gene(s) of
  interest to that of an endogenous control gene.
  Quantitation is done relative to the control gene.

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Common Uses

• Microarray/Differential Expression
• RNAi
• Diagnostics
• SNP Genotyping

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Differential Expression

• Compares transcriptional levels of genes between
  control and experimental samples
• Endogenous controls are used to normalize the
  data
• Endogenous controls are genes common to both
  control and experimental samples that do not
  change their expression levels under the
  experimental conditions
• GAPDH
• ß-Actin
• 18s ribosomal subunit

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Threshold Cycle

• As the PCR progresses into the exponential phase,
  the system detects a cycle when the fluorescence
  detected is significantly higher than the
  baseline value.
• Threshold cycle (Ct) point at which the
  fluorescent signal is first recorded as
  statistically significant above background
• The Ct value of each RT-PCR reaction depends on
  the initial template amount (copy number) of the
  target sequence, and it is inversely proportional
  to the log of this copy number.
• The more templates present at the beginning of
  the reaction, the fewer number of cycles it takes
  to reach Ct.
• Real-time PCR now makes quantitation of DNA and
  RNA much more precise and reproducible because it
  relies on Ct values determined during the
  exponential phase of PCR rather then endpoint.

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Comparing Ct Values
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RNAi

• RNA interference double stranded RNA (siRNA)
  forms a complex (RISC) that binds to target mRNA
  and leads to its degradation

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Diagnostics

• Use a panel of genes representative of conditions
  in question
• Presence of certain genes in the panel can be
  used to identify specific conditions

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SNP

• Single Nucleotide Polymorphisms
• Allelic Discrimination Assays
• Genomic locus where two or more alternative bases
  occur with appreciable frequency