Analysis of gene expression by
real-time PCR
RBCS3 and Cab-1b transcript
quantitation by real time PCR
Broad and Long Term Objective
To characterize the expression of the genes encoding
Ribulose 1-5 bisphosphate carboxylase oxygenase-3
(RBCS3) and Chlorophyll A/B binding protein-1b (Cab1b) in the leaves of tomato plants grown under
continuous light or subjected to 24 hr, 48 hr, or 72 hr in
complete darkness.
Research Plan
RNA Isolation from tomato leaves (continuous light or
24-72 hr darkness)
RNA Electrophoresis
cDNA synthesis
RBCS3 and Cab-1b transcript quantitation
by real time PCR
Analysis of real time PCR data
Today’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
PCR
• 10X buffer
• Primers
• Taq DNA
polymerase
• dNTPs
• template DNA
After 30 thermocycles,
a DNA molecule initially
present in a single copy
is present in > 1 billion
copies
Standard PCR is non-quantitative
Primers/dNTPs exhausted
DNA amount (arbitrary units)
100
electrophoresis
Post-PCR DNA
quantitation
0
15
PCR cycle #
White tube= 1000 copies
Green tube= 10 copies
30
Equal amounts
of PCR product
Real time PCR is quantitative
100
Fluorescence (arbitrary units)
White tube= 1000 copies
Green tube= 10 copies
Ct = 10.0
• 10X buffer
• Primers
• Taq DNA
polymerase
• dNTPs
• template DNA
• fluorescent
“marker” of
dsDNA
Ct = 16.6
6.6
Fluorescence
threshold
0
15
“real time” DNA quantitation
30
16.6-10.0 = 6.6 cycles
26.6 = 100
White sample is
100-fold more abundant
than green sample
Fluorescence (arbitrary units)
Output data from real time PCR
PCR cycle #
Fluorescence-based chemistries
for 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
Fluorescence-based chemistries
for quantitation of dsDNA
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
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 hardware
Optical quality
PCR tubes or
96 well plate
BioRad icycler
excitation
emission
*
Illumination: laser, LED,
tungsten halogen bulb
Light filter
Detector: photomultiplier
Experimental Design
Time Series
►
►
RNA Extractions Performed on tomato plants grown in the light or in
darkness for 24, 48, or 72 hrs
Each sample assayed in triplicate in 96-well format
Reaction Mix
cDNA (diluted 1:100)
Sybr Green Super Mix
Primer RB1 or CA1 (20 μM)
Primer RB2 or CA2 (20 μM)
dH2O
total volume per reaction
5.0 μl
7.1 μl
0.3 μl
0.3 μl
12.3 μl
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
Parameters
Polymerase activation
95° C
10 min
40 cycles
Denaturation
Primer Annealing
Extension
95° C
60° C
72° C
10 sec
30 sec
30 sec
Melt Curves
Denaturation
Renaturation
Denaturation
95° C 1 min
55° C 1 min
Ramp 0.5° C every 10 sec
Alternative methods for transcript
quantitation
Northern Blot
RNase protection assay
Next Week
Analysis of real time PCR data
NDB2
4.5
4.0
3.5
3.0
Co
2.5
2.0
Ni
Am
1.5
1.0
0.5
0.0
Leaf
Root
RNA gels
RNA ladder
Class gels
Expected result (plant RNA)