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Analysis of gene expression by Analysis of gene expression by real-time PCRreal-time PCR
RBCS3RBCS3 and and Cab-1bCab-1b transcript transcript
quantitation by real time PCR quantitation by real time PCR
Broad and Long Term ObjectiveBroad and Long Term Objective
To characterize the expression of the genes encoding To characterize the expression of the genes encoding Ribulose 1-5 bisphosphate carboxylase oxygenase-3 Ribulose 1-5 bisphosphate carboxylase oxygenase-3 ((RBCS3RBCS3) and Chlorophyll A/B binding protein-1b () and Chlorophyll A/B binding protein-1b (Cab-Cab-1b1b) in the leaves of tomato plants grown under ) in the leaves of tomato plants grown under continuous light or subjected to 24 hr, 48 hr, or 72 hr in continuous light or subjected to 24 hr, 48 hr, or 72 hr in complete darkness.complete darkness.
Research PlanResearch Plan
RNA Electrophoresis
RNA Isolation from tomato leaves (continuous light or 24-72 hr darkness)
cDNA synthesis
RBCS3 and Cab-1b transcript quantitation by real time PCR
Analysis of real time PCR data
Today’s Laboratory ObjectivesToday’s Laboratory Objectives
1.To set up and run a series of real time PCR reactions with Cab-1b- and RBCS3-specific primers
2.To understand the theoretical basis of real time PCR and its quantitative applications
PCRPCR
• 10X buffer• Primers• Taq DNA polymerase• dNTPs• template DNA
After 30 thermocycles,a DNA molecule initiallypresent in a single copyis present in > 1 billion copies
Standard PCR is non-quantitativeStandard PCR is non-quantitative
PCR cycle #
0 15 30
DN
A a
mou
nt (
arbi
trar
y un
its)
100
White tube= 1000 copiesGreen tube= 10 copies
Primers/dNTPs exhausted
electrophoresis
Equal amounts of PCR product
Post-PCR DNA quantitation
Real time PCR is quantitativeReal time PCR is quantitative
0 15 30
Flu
ores
cenc
e (a
rbitr
ary
units
)
100
6.6
16.6-10.0 = 6.6 cycles
26.6 = 100
White sample is100-fold more abundantthan green sample
“real time” DNA quantitation
Fluorescence threshold
• 10X buffer• Primers• Taq DNA polymerase• dNTPs• template DNA• fluorescent “marker” of dsDNA
Ct = 10.0Ct = 16.6
White tube= 1000 copiesGreen tube= 10 copies
Output data from real time PCROutput data from real time PCR
PCR cycle #
Flu
ores
cenc
e (a
rbitr
ary
units
)
Fluorescence-based chemistries Fluorescence-based chemistries for quantitation of dsDNAfor quantitation of dsDNA
SYBR green I• cyanine dye (abs. 480nm, em. 520nm)• binds almost exclusively to dsDNA (minor groove)• fluorescence increases >1000-fold when bound to DNA• sensitive, but nonspecific
TaqMan• 15-30 bp ssDNA probe, complementary to template DNA sequence• 5’ fluorescent dye, 3’ fluorescence quencher• Taq 5’ exonuclease activity cleaves ssDNA probe, releasing free dye into solution (fluorescence)• sensitive, sequence specific, multiplex PCR possible
Molecular beacons• 40-50 bp ssDNA probe, the central region of sequence is complementary to template DNA• 5’ fluorescent dye, 3’ fluorescence quencher• 5’ and 3’ end of sequence are self complementary, form stem loop structure which promotes fluorescence quenching• during annealing phase of PCR, the central part of the probe hybridizes with the complementary template DNA sequence, separating fluorescent dye and quencher, resulting in fluorescence
Fluorescence-based chemistries Fluorescence-based chemistries for quantitation of dsDNAfor quantitation of dsDNA
For all chemistries, fluorescence and dsDNA content are directly proportional(i.e. fluorescence doubles after each thermocycle). Therefore, DNA content in the PCR reaction can be quantified after each cycle by measuring fluorescence
Real time PCR hardwareReal time PCR hardware
Optical qualityPCR tubes or96 well plate
BioRad icycler
Illumination: laser, LED,tungsten halogen bulb
excitation emission
*Light filter
Detector: photomultiplier
Experimental DesignExperimental Design
Time SeriesTime Series► RNA Extractions Performed on tomato plants grown in the light or in RNA Extractions Performed on tomato plants grown in the light or in
darkness for 24, 48, or 72 hrsdarkness for 24, 48, or 72 hrs► Each sample assayed in triplicate in 96-well formatEach sample assayed in triplicate in 96-well format
Reaction MixcDNA (diluted 1:100) 5.0 μlSybr Green Super Mix 7.1 μlPrimer RB1 or CA1 (20 μM) 0.3 μlPrimer RB2 or CA2 (20 μM) 0.3 μldH2O 12.3 μltotal volume per reaction 25 μl
8 total reactions per group (e.g. 3 RB1/RB2 + light cDNA, 3 RB1/RB2 + dark cDNA, 2 RB1/RB2 + water)
Real Time RT PCR Cycling Real Time RT PCR Cycling ParametersParameters
Polymerase activationPolymerase activation 95° C 95° C 10 min10 min
40 cycles40 cyclesDenaturationDenaturation 95° C 95° C 10 sec10 secPrimer AnnealingPrimer Annealing 60° C 60° C 30 sec30 secExtensionExtension 72° C 72° C 30 sec30 sec
Melt CurvesMelt CurvesDenaturationDenaturation 95° C 95° C 1 min1 minRenaturationRenaturation 55° C 55° C 1 min1 minDenaturationDenaturation Ramp 0.5° C every 10 secRamp 0.5° C every 10 sec
Alternative methods for transcript Alternative methods for transcript quantitationquantitation
Northern Blot RNase protection assay
Next WeekNext Week
Analysis of real time PCR data
NDB2
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
4.0
4.5
Leaf Root
Co
Ni
Am