Realtime

Pathway-Centric Tools and Technology™

Using Real-Time & End-Point PCR for Gene Expression Analysis and

Array Data Verification

SuperArray Bioscience CorporationGeorge J. Quellhorst, Jr. Ph.D.Manager, Customer Education


Topics to be Covered

Real-Time PCRIntroduction, SYBR Green DetectionConsiderations for SuccessExamples of Dynamic Range & Specificity

End-Point (Conventional) PCRIntroduction, Considerations for SuccessMore Quantitative with Internal NormalizerDynamic Range, Control Systematic Variation

Examples of Expression Profiling for both


Real-Time PCR

Monitors amount of amplicon DURING reactionAmplicon amount DIRECTLY related to fluorescenceGene expression INVERSELY PROPORTIONAL to CtVERY quantitativeWIDE dynamic range and HIGH sensitivityDOES NOT require post-reaction processingREQUIRES dedicated equipmentCAN BE expensive, but not necessarily


SYBR® Green Detection

Fluoresces when bound to dsDNAAmplicon detected either during annealing step or at the end of the extension stepDetects any dsDNA non-specificallyLeast expensive & easiest to useApplicable to most real-time systems


SYBR® Green Detection

Melting

Extension

MeltingAnnealing


Considerations for Successful Real-Time PCR:Primer Design

High Amplification EfficiencyShort amplicon size (100 to 200 bp)

SpecificityUniqueness in the genome – especially at 3’ endHigh sequence complexityNo secondary structure or primer dimersOnly one gene-specific amplicon detectable

Chemical PropertiesMelting temperature / GC content


Considerations for Successful Real-Time PCR:Master Mix and Others

HotStart enzymeHighly proficient enzyme = high amplification efficiencyMinimizes amplification of non-specific priming eventsFurther insures only one amplicon

Wide Dynamic RangeFive to six orders of magnitude

Accommodation of SYBR® Green DetectionConvenient: If lyophilized, just add water.


Five-Log Dynamic Range

y = -13.73Lg(x) + 18.745 R2 = 0.9957

05

10152025303540

0.00001 0.0001 0.001 0.01 0.1 1Dilution factor

Thre

shol

d cy

cle

cDNA

0

10-4

10-3

10-2

10-1

1

10-5

** 1X cDNA = Product (1 µl) of 25-µl First Strand cDNA Synthesis using 0.15 µg Universal Reference RNA

cDNA 0 10-5 10-4 10-3 10-2 10-1 1**


High Specificity: Single PCR ProductsFirst Derivative Melting Curves

BMP1 BMP2 BMP3 BMP4

BMP5 BMP6 BMP7 BMP15

Failed Primer DesignBMP1 BMP2 BMP3 BMP4 BMP5 BMP6 BMP7 BMP15

Agarose Gel

RT2 Real-Time™ Gene ExpressionAssay Kits for BMP family


Gene Expression Profiling with Real-Time PCR:Generating Standard Curve

0

5

10

15

20

25

30

0.001 0.01 0.1 1

Dilution factor

Thre

shol

d C

ycle

GAPDCt=-3.351Log(x)+13.4

TNFAIP3 Ct=-3.302Log(x)+16.612

Serial Dilution ofXpressRef™ Human Universal Reference

RNA (GA-004)

First Strand cDNA Synthesis Kit(Cat # C-01)

RT2 Real-Time™Gene Expression Assay Kit


Gene Expression Profiling with Real-Time PCR:Generating Standard Curve

05

1015202530

0.001 0.01 0.1 1

Dilution Factor

Thre

shold

Cycle

0.0048 /0.17 /(TNFAIP3/GAPD)treated

(TNFAIP3/GAPD)untreated

=

GAPD Internal Normalizer

TNFAIP3

untreated TNFα treated

0.130.17

0.00480.14

TNFα untreated: Ct(TNFAIP3)=24.25, Ct(GAPD)=16.49TNFα treated: Ct(TNFAIP3)=19.17, Ct(GAPD)=16.36

0.130.14

RNA from HeLa CellsTreated ± TNFα

First Strand cDNA Synthesis Kit(Cat # C-01)

RT2 Real-Time™Gene Expression Assay Kit

= 32.9 fold-change inTNFAIP3 expression


Gene Expression Profiling with Real-Time PCR:Quantification by ∆∆Ct**

If the PCR efficiency is 1 (if the calibration curve slopes m equal -3.3), then the fold-change in gene expression = 2-∆∆Ct.

GAPD: Ct=-3.351Lg(x)+13.4TNFAIP3: Ct=-3.302Lg(x)+16.612 Standard curves:

TNFα untreated: Ct(TNFAIP3)=24.25 Ct(GAPD)=16.49TNFα treated: Ct(TNFAIP3)=19.17 Ct(GAPD)=16.36

∆Ct (treated) = Ct (TNFAIP3) - Ct (GAPD) = 19.17 – 16.36 = 2.81∆Ct (untreated) = Ct (TNFAIP3) - Ct (GAPD) = 24.25 – 16.49 = 7.76∆∆Ct = ∆Ct (treated) - ∆Ct (untreated) = 2.81 – 7.76 = -4.95

The fold-change in TNFAIP3 expression = 2-∆∆Ct = 24.95 = 30.9Error = |(30.9- 32.9)|/32.9 = 6%

** Use this method ONLY if the replication efficiencies for your gene of interest and the housekeeping gene are the same or similar.


Gene Expression Profiling with Real-Time PCR:Quantification by ∆∆Ct**

If the PCR efficiency is less than 1,then the fold-change in gene expression = 10∆∆Ct/m.

GAPD: Ct=-3.351Lg(x)+13.4TNFAIP3: Ct=-3.302Lg(x)+16.612 Standard curves:

Average slope m = ½ (- 3.302 - 3.351) = -3.327

∆∆Ct = ∆Ct (treated) - ∆Ct (untreated) = -4.95

The fold-change in TNFAIP3 expression = 10∆∆Ct/m = 10-4.95/-3.327 = 30.8

Error = |(30.8- 32.9)|/32.9 = 6%

** Use this method ONLY if the replication efficiencies for your gene of interest and the housekeeping gene are the same or similar.


Real-Time PCR Summary:With proper primer design,

SYBR Green detection applicable to all genesGene-specific ampliconsFive-fold dynamic rangeSimple determination of relative gene expressionUseful for direct expression profiling and array data verification on gene-by-gene basis


RT2 Real-Time™Gene Expression Assay Kits

Available for any human, mouse, or rat geneExperimentally verified computer algorithm primer designOptimized for SYBR® Green detectionIncludes gene-specific primer pair & PCR master mix


End-Point (Conventional) PCR

Determines amount of amplicon AFTER reactionAmplicon amount NOT DIRECTLY related to intensityNOT AS quantitative, but can be improvedNARROWER dynamic range and LOWER sensitivityREQUIRES post-reaction processingDOES NOT require special equipmentLESS expensive


Considerations for Successful End-Point PCR

PrimersGene-specific amplicons

As Wide a Dynamic Range as possibleTwo to three orders of magnitude

Convenient Master MixIf lyophilized, just dissolve.Including gel loading dye saves a step.

Mechanism to normalize resultsInternal Normalizer for housekeeping gene



Makes Conventional RT-PCR MORE quantitative.Detects the relative GAPD expression level in same tube as the gene of interest.Attenuates GAPD Signal.

Band intensity does not saturate.Does not interfere with detection of the gene of interest.Places GAPD detection in same dynamic range as most genes.

Relative Gene Expression = Target-to-GAPD RatioRatios compared between experimental conditions to control for systematic variables.


GAPD Internal Normalizer Corrects for Differences in Template Loading

First Strand cDNASynthesis Kit(Cat # C-01)

Standard GAPD PrimersHuman MX1 and IRF7

RT2 End-Point™ Kits

XpressRef™ HumanUniversal ReferenceTotal RNA (GA-004)

cDNA 1 µL 2 µL 1 µL 2 µL

Standard GAPD Primers

MX1 (222 bp)

GAPD orInternalNormalizer(436 bp)

IRF7 (163 bp) 0.58 0.50 N/A N/A

Target/GAPDH


GAPD Internal Normalizer Corrects for Differences in Template Loading

Standard or Internal NormalizerGAPD Primers

Human MX1 and IRF7


First Strand cDNASynthesis Kit(Cat # C-01)XpressRef™ Human

Universal ReferenceTotal RNA (GA-004)


Standard GAPD Primers

0.58 0.50 N/A N/ATarget/GAPDH

MX1 (222 bp)

GAPD orInternalNormalizer(436 bp)

IRF7 (163 bp)



1.1 1.1 0.40 0.41Target/GAPDH


Dynamic Range of End-Point RT-PCRUsing Internal Normalizer

First Strand cDNASynthesis Kit (Cat # C-01)XpressRef™ Human

Universal Reference Total RNA(GA-004)

cDNA

Spike withHuman PARP-1 plasmid RT2 End-Point™ Kit

Human PARP1cDNA

Human GAPD Internal Normalizer

PARP-1

cDNA 1µL 1µL 1µL 1µL 1µL 1µL 1µL 1µL

PARP-1 2x106 106 5x105 2.5x105 105 6x104 3x104 0 copies


Dynamic Range of End-Point RT-PCRUsing Internal Normalizer - continued

y = 2E-06x R2 = 0.9826

00.10.20.30.40.50.60.70.80.9

0 100000 200000 300000 400000 500000 600000Copies of Target

Hum

an P

AR

P1:

GA

PD

Rat

io


Expression Profiling with End-Point PCR and Internal Normalizer

First Strand cDNASynthesis Kit(Cat # C-01)RNA from HT-29 Cells

Treated ± IFNα(10 ng/ml 3h 37C)

Human MX1 and IRF7


MX1 (414 bp)GAPD Internal Normalizer (226 bp)

1000bp750bp500bp

250bp

IFN-α- +MX1

GAPD Internal Normalizer (226 bp)

IRF7 (315 bp)

1000bp750bp500bp

250bp

IFN-α- +IRF7

IRF7 / GAPD

MX1 / GAPD

Fold IncreaseIFNαUntreated

2.440.61 4.0

0.79 4.60.17


Expression Profiling with RT2 End-Point™ Gene Expression Assay Kits

RNA from HeLa CellsTreated with TNFαFor various times

First Strand cDNASynthesis Kit(Cat # C-01) Human TNFAIP3, NFKBIA, IL6


30min 1hr 2hr 3hr 4hruntreated

TNFα treated

Target/GAPD 0.41 0.98 1.5 1.3 1.4 1.1

Target/GAPD 0.13 0.77 1.5 1.5 0.81 1.0

Target/GAPD 0.30 2.3 2.3 1.2 0.90 1.0

GAPD

TNFAIP3

NFKBIA

GAPD

IL6

GAPD

0.0

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5.0

7.5

10.0

12.5

15.0

0 1 2 3 4Induction time (hrs)

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TNFAIP3NFKBIAIL6


End-Point PCR SummaryWith use of an Internal Normalizer,

Provides adequate dynamic rangesControls for systematic tube-to-tube variationSimple determination of relative gene expressionUseful for direct expression profiling and array data verification on gene-by-gene basisProvides quantitative RT-PCR to researchers without access to real-time equipment


RT2 End-Point™ Gene Expression Assay Kits

Includes enough reagents for 24 reactions:Primer pair designed with experimentally-verified computer algorithmHotStart “Sweet ” PCR master mixUnique GAPD Internal Normalizer

Band of correct size from universal RNA sourceAny gene in the human, mouse, or rat genome


Using Real-Time & End-Point PCR for Gene Expression Analysis and

Array Data Verification

SuperArray Bioscience CorporationGeorge J. Quellhorst, Jr. Ph.D.Manager, Customer Education

Realtime

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