Real Time RTPCR qPCR

1
Effect of increasing extracellular Ca2 levels
on intracellular Ca2 fluxes in lily pollen tubes
Aequorin luminescence

• Extracellular calcium changed from 0.13 mM to 1.3
  mM
• Extracellular calcium changed from 1.3 mM to 10
  mM
• Extracellular calcium changed from 10 mM to 0.13
  mM

Messerli and Robinson, Planta 2003
2
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

3
The Lux (Light Upon eXtension) effect
4
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

5
Steps for Starting qPCR

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

6
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

7
Quantitative Real-time PCR analysis of Annexin
T-DNA insertion lines
Relative Expression
WT
WT
WT
WT
WT
WT
AnnAt 6-2
AnnAt 6-1
AnnAt 5-1
AnnAt 5-2
AnnAt 1-1
AnnAt 1-3
AnnAt 2-2
AnnAt 2-1
AnnAt 3-1
AnnAt 3-2
AnnAt 1-2
Flowers
AnnAt 4-2
AnnAt 4-1
8
Quantitative Real-time PCR analysis of Annexin
T-DNA insertion lines
Relative Expression
WT
WT
WT
WT
WT
WT
7-day old seedlings
AnnAt 1-2
AnnAt 1-1
AnnAt 3-1
AnnAt 3-2
AnnAt 6-2
AnnAt 6-1
AnnAt 5-2
AnnAt 7-1
AnnAt 8-1
9
Quantitative Real-time PCR analysis of AnnAt7
T-DNA insertion line
Relative Expression
AnnAt 7-2
WT
10
Quantitative Real-time PCR analysis of AnnAt8
T-DNA insertion line
Relative Expression
WT
AnnAt 8-2