Quantitative Real Time RTPCR qPCR

1
Quantitative Real Time RT-PCR (qPCR)

• Use of fluorescence in PCR reactions to monitor
  the production of target at each step
• Applications for
• Comparing relative changes in RNA levels
  (treatment conditions, mutants or time)
• Calculating RNA copy number
• Single nucleotide polymorphism detection

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The Lux (Light Upon eXtension) effect
3
Cycle Threshold (Ct)

• The cycle threshold is the cycle at which the
  measured fluorescence (and therefore product)
  reaches a certain level
• As starting template increases, the Ct decreases

4
Steps for Starting qPCR

• Choose and test control genes
• Experimentally determine optimal primer
  concentrations
• Compare amplification efficiencies
• Run experiment
• For new genes, compare efficiencies

5
Control Gene Requirements

• Constant expression levels over experimental
  conditions (time or treatments)
• i. e. housekeeping genes
• Similar amplification efficiencies to
  experimental gene
• For Arabidopsis used adenosine phosphoribosyltrans
  ferase 1

6
Quantitative Real-time PCR analysis of Annexin
T-DNA insertion lines
7
Relative expression of Arabidopsis annexins in
seeds during germination
8
Western blot analyses of Arabidopsis plasma
membrane fractions
1249 AnnAt2 1250 AnnAt1 E8 pea p35
9
Genome-wide non-mendelian inheritance of
extra-genomic information in Arabidopsis Susan
J. Lolle, Jennifer L. Victor, Jessica M. Young
Robert E. Pruitt NATURE VOL 434 24 MARCH
2005 pp505-509