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

RNA gelsRNA gels

RNA ladder Class gels Expected result (plant RNA)