1. Pathway-Centric Tools and Technology™
Using Real-Time & End-Point PCR
for Gene Expression Analysis and
Array Data Verification
SuperArray Bioscience Corporation
George J. Quellhorst, Jr. Ph.D.
Manager, Customer Education

2. Pathway-Centric Tools and Technology™
Topics to be Covered
Real-Time PCR
Introduction, SYBR Green Detection
Considerations for Success
Examples of Dynamic Range & Specificity
End-Point (Conventional) PCR
Introduction, Considerations for Success
More Quantitative with Internal Normalizer
Dynamic Range, Control Systematic Variation
Examples of Expression Profiling for both

3. Pathway-Centric Tools and Technology™
Real-Time PCR
Monitors amount of amplicon DURING reaction
Amplicon amount DIRECTLY related to fluorescence
Gene expression INVERSELY PROPORTIONAL to Ct
VERY quantitative
WIDE dynamic range and HIGH sensitivity
DOES NOT require post-reaction processing
REQUIRES dedicated equipment
CAN BE expensive, but not necessarily

4. Pathway-Centric Tools and Technology™
SYBR® Green Detection
Fluoresces when bound to dsDNA
Amplicon detected either during annealing step
or at the end of the extension step
Detects any dsDNA non-specifically
Least expensive & easiest to use
Applicable to most real-time systems

5. Pathway-Centric Tools and Technology™
SYBR® Green Detection
Melting
Melting
Annealing
Extension

6. Pathway-Centric Tools and Technology™
Considerations for Successful Real-Time PCR:
Primer Design
High Amplification Efficiency
Short amplicon size (100 to 200 bp)
Specificity
Uniqueness in the genome – especially at 3’ end
High sequence complexity
No secondary structure or primer dimers
Only one gene-specific amplicon detectable
Chemical Properties
Melting temperature / GC content

7. Pathway-Centric Tools and Technology™
Considerations for Successful Real-Time PCR:
Master Mix and Others
HotStart enzyme
Highly proficient enzyme = high amplification efficiency
Minimizes amplification of non-specific priming events
Further insures only one amplicon
Wide Dynamic Range
Five to six orders of magnitude
Accommodation of SYBR® Green Detection
Convenient: If lyophilized, just add water.

8. Pathway-Centric Tools and Technology™
Five-Log Dynamic Range
cDNA
0
10-5
10-4
10-3
10-2
10-1
1
Threshold cycle
y = -13.73Lg(x) + 18.745 R2 = 0.9957
40
35
30
25
20
15
10
5
0
0.00001
cDNA
0
10-5
10-4
10-3 10-2
10-1
1**
** 1X cDNA = Product (1 µl) of 25-µl First
Strand cDNA Synthesis using 0.15 µg
Universal Reference RNA
0.0001
0.001
0.01
Dilution factor
0.1
1

9. Pathway-Centric Tools and Technology™
High Specificity: Single PCR Products
First Derivative Melting Curves
BMP1
BMP2
BMP4
BMP6
BMP5
BMP3
BMP7
BMP15
Failed Primer Design
Agarose Gel
BMP1 BMP2 BMP3
BMP4 BMP5
BMP6 BMP7 BMP15
RT2 Real-Time™ Gene Expression
Assay Kits for BMP family

10. Pathway-Centric Tools and Technology™
Gene Expression Profiling with Real-Time PCR:
Generating Standard Curve
First Strand cDNA Synthesis Kit
Serial Dilution of
(Cat # C-01)
XpressRef™ Human
Universal Reference
RNA (GA-004)
RT2 Real-Time™
Gene Expression Assay Kit
Threshold Cycle
30
25
TNFAIP3
Ct=-3.302Log(x)+16.612
20
15
GAPD
Ct=-3.351Log(x)+13.4
10
5
0
0.001
0.01
0.1
Dilution factor
1

11. Pathway-Centric Tools and Technology™
Gene Expression Profiling with Real-Time PCR:
Generating Standard Curve
First Strand cDNA Synthesis Kit
(Cat # C-01)
RNA from HeLa Cells
Treated ± TNFα
RT2 Real-Time™
Gene Expression Assay Kit
TNFα untreated: Ct(TNFAIP3)=24.25, Ct(GAPD)=16.49
TNFα treated: Ct(TNFAIP3)=19.17, Ct(GAPD)=16.36
Threshold Cycle
30
(TNFAIP3/GAPD)treated
25
=
(TNFAIP3/GAPD)untreated
20
0.17 / 0.14
0.0048 / 0.13
= 32.9 fold-change in
TNFAIP3 expression
15
10
5
untreated
0
0.001
0.01
0.0048
0.1
Dilution Factor
0.17
0.13
0.14
1
TNFα treated
TNFAIP3
GAPD
Internal
Normalizer

12. Pathway-Centric Tools and Technology™
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.
Standard curves: TNFAIP3: Ct=-3.302Lg(x)+16.612
GAPD: Ct=-3.351Lg(x)+13.4
TNFα untreated: Ct(TNFAIP3)=24.25
TNFα treated: Ct(TNFAIP3)=19.17
Ct(GAPD)=16.49
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 = 30.9
Error = |(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.

13. Pathway-Centric Tools and Technology™
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.
Standard curves: TNFAIP3: Ct=-3.302Lg(x)+16.612
GAPD: Ct=-3.351Lg(x)+13.4
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.

14. Pathway-Centric Tools and Technology™
Real-Time PCR Summary:
With proper primer design,
SYBR Green detection applicable to all genes
Gene-specific amplicons
Five-fold dynamic range
Simple determination of relative gene expression
Useful for direct expression profiling and array data
verification on gene-by-gene basis

15. Pathway-Centric Tools and Technology™

16. Pathway-Centric Tools and Technology™
RT2 Real-Time™
Gene Expression Assay Kits
Available for any human, mouse, or rat gene
Experimentally verified computer algorithm primer design
Optimized for SYBR® Green detection
Includes gene-specific primer pair & PCR master mix

17. Pathway-Centric Tools and Technology™
End-Point (Conventional) PCR
Determines amount of amplicon AFTER reaction
Amplicon amount NOT DIRECTLY related to intensity
NOT AS quantitative, but can be improved
NARROWER dynamic range and LOWER sensitivity
REQUIRES post-reaction processing
DOES NOT require special equipment
LESS expensive

18. Pathway-Centric Tools and Technology™
Considerations for Successful End-Point PCR
Primers
Gene-specific amplicons
As Wide a Dynamic Range as possible
Two to three orders of magnitude
Convenient Master Mix
If lyophilized, just dissolve.
Including gel loading dye saves a step.
Mechanism to normalize results
Internal Normalizer for housekeeping gene

19. Pathway-Centric Tools and Technology™
GAPD Internal Normalizer
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 Ratio
Ratios compared between experimental conditions to control for
systematic variables.

20. Pathway-Centric Tools and Technology™
GAPD Internal Normalizer Corrects for
Differences in Template Loading
First Strand cDNA
Synthesis Kit
XpressRef™ Human
(Cat # C-01)
Universal Reference
Total RNA (GA-004)
Standard GAPD Primers
Human MX1 and IRF7
RT2 End-Point™ Kits
Standard GAPD Primers
cDNA 1 µL
2 µL 1 µL
2 µL
GAPD or
Internal
Normalizer
(436 bp)
MX1 (222 bp)
0.58
0.50 N/A N/A
Target/GAPDH
IRF7 (163 bp)

21. Pathway-Centric Tools and Technology™
GAPD Internal Normalizer Corrects for
Differences in Template Loading
First Strand cDNA
Synthesis Kit
XpressRef™ Human
(Cat # C-01)
Universal Reference
Total RNA (GA-004)
Standard GAPD Primers
cDNA 1 µL
2 µL 1 µL
2 µL
Standard or Internal Normalizer
GAPD Primers
Human MX1 and IRF7
RT2 End-Point™ Kits
GAPD Internal Normalizer
cDNA
1 µL
2 µL
1 µL
2 µL
GAPD or
Internal
Normalizer
(436 bp)
MX1 (222 bp)
0.58
0.50 N/A N/A
Target/GAPDH
1.1
1.1
0.40 0.41
Target/GAPDH
IRF7 (163 bp)

22. Pathway-Centric Tools and Technology™
Dynamic Range of End-Point RT-PCR
Using Internal Normalizer
XpressRef™ Human
Universal Reference Total RNA
(GA-004)
cDNA
cDNA
First Strand cDNA
Synthesis Kit (Cat # C-01)
Spike with
Human PARP-1 plasmid
cDNA
RT2 End-Point™ Kit
Human PARP1
1µL
1µL
PARP-1 2x106 106
1µL
1µL
1µL
5x105 2.5x105 105
1µL
1µL
6x104 3x104
1µL
0 copies
PARP-1
Human GAPD
Internal Normalizer

23. Pathway-Centric Tools and Technology™
Dynamic Range of End-Point RT-PCR
Using Internal Normalizer - continued
Human PARP1:GAPD Ratio
y = 2E-06x R2 = 0.9826
0.9
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0
0
100000
200000
300000
400000
Copies of Target
500000
600000

24. Pathway-Centric Tools and Technology™
Expression Profiling with
End-Point PCR and Internal Normalizer
First Strand cDNA
Synthesis Kit
RNA from HT-29 Cells
(Cat # C-01)
Treated ± IFNα
(10 ng/ml 3h 37C)
MX1
IFN-α
+
1000bp
750bp
500bp
IRF7
MX1 (414 bp)
250bp
GAPD Internal
Normalizer (226 bp)
Human MX1 and IRF7
RT2 End-Point™ Kits
IFN-α
+
1000bp
750bp
500bp
250bp
Untreated
IFNα
Fold Increase
MX1 / GAPD
0.61
2.44
4.0
IRF7 / GAPD
0.17
0.79
4.6
IRF7 (315 bp)
GAPD Internal
Normalizer (226 bp)

25. Pathway-Centric Tools and Technology™
Expression Profiling with
RT2 End-Point™ Gene Expression Assay Kits
First Strand cDNA
Synthesis Kit
RNA from HeLa Cells
(Cat # C-01)
Treated with TNFα
For various times
15.0
TNFα treated
30min
1hr
2hr
3hr
4hr
GAPD
TNFAIP3
Target/GAPD
0.41
0.98
1.5
1.3
1.4
1.1
GAPD
NFKBIA
Target/GAPD
0.13
0.77
1.5
1.5
0.81
1.0
GAPD
IL6
Target/GAPD
0.30
RT2 End-Point™ Kits
2.3
2.3
1.2
0.90
1.0
Induction level
untreated
Human TNFAIP3, NFKBIA, IL6
TNFAIP3
NFKBIA
IL6
12.5
10.0
7.5
5.0
2.5
0.0
0
1
2
3
Induction time (hrs)
4

26. Pathway-Centric Tools and Technology™
End-Point PCR Summary
With use of an Internal Normalizer,
Provides adequate dynamic ranges
Controls for systematic tube-to-tube variation
Simple determination of relative gene expression
Useful for direct expression profiling and array data
verification on gene-by-gene basis
Provides quantitative RT-PCR to researchers without
access to real-time equipment

27. Pathway-Centric Tools and Technology™
RT2 End-Point™ Gene Expression Assay Kits
Includes enough reagents for 24 reactions:
Primer pair designed with experimentally-verified
computer algorithm
HotStart “Sweet ” PCR master mix
Unique GAPD Internal Normalizer
Band of correct size from universal RNA source
Any gene in the human, mouse, or rat genome

28. Pathway-Centric Tools and Technology™
Using Real-Time & End-Point PCR
for Gene Expression Analysis and
Array Data Verification
SuperArray Bioscience Corporation
George J. Quellhorst, Jr. Ph.D.
Manager, Customer Education