1. Real-Time quantitative PCR: Choices and Decisions
Dr Sandrine Javorschi-Miller
R&D program Manager
European Functional Genomic Seminar
May 2005

2. Why using real-time PCR?

3. Gene Expression Analysis Technologies
>10,000
Real-time PCR
100
Low-density
Arrays
Number of samples 10
High-density
Arrays
RPA
Northern
SAGE 1
2-10
>10,000
Number of genes
To consider: Ratio samples / genes, needs for accuracy

4. Comparison of Quantitative Assays (RNA/DNA):
Sensitivity
Dynamic Range
Real-Time PCR
Amplicor/TMA NASBA
bDNA
XPLORE
Microarrays
RPA
Northern
100
101
102
103
104
105
106
107
108
108
107
106
105
104
103
102
101
100
NASBA: nucleic acid seq based amplification
bDNA: branched DNA assay
Xplore: based on Invader technology
TMA: transcription mediated amplification
RPA: RNAse protection assay
Advantage: Real-Time PCR

5. Real-time PCR in more details

6. Phases of amplification
Exponential phase
Plateau phase

7. Exponential phase vs. plateau
At some time or another, all reactions regardless of initial amount reach the same plateau!
Plateau is not quantitative
Exponential phase is quantitative
Cycles
Amplicon
amount
Plateau information is not quantitative

8. Before the Real-Time era: End-point PCR
Examples of semi-quantitative PCR
End point analysis after reduce number of cycles (exponential phase)
Not accurate
Not sensitive
Competitive PCR
Time and reagent consuming
GOI (pg)
0.1 1 10 ?
PCR condition
95oC for 2 min
# of cycles oC for 15 sec
62oC for 30 sec
72oC for 30 sec
End Point is at best semi-quantitative

9. Choices and Decisions

10. One Step or Two Step RT-PCR?

11. One Step or Two Step RT-PCR?
One Step RT-PCR
(One tube)
Two Step RT-PCR
(Two tubes)
RNA
1 target
X targets
Oligo dT
Random Primers
(GS Primers)
GS primers
cDNA
Target pool
1 amplicon
Amplicon
Amplicon
X amplicons

12. Two-step RT-PCR is more convenient and cost effective
One Step or Two Step RT-PCR?
One-Step RT-PCR
Highly defined conditions to support RT and Taq
Requires gene specific primer
Ideal for quantification of 1 or 2 messages from a large number of RNA samples
Two-Step RT-PCR
Separate conditions for cDNA synthesis & PCR
Flexible choice of primers
Ideal for quantification of multiple genes from a limited number of RNA samples
Perfect for:
- Lot of samples
Small amount of targets
Perfect for:
- Few samples
- Large amount of targets
Two-step RT-PCR is more convenient and cost effective

13. Which Reverse Transcriptase?

14. Good Reverse transcriptase is essential!
What RT enzyme?
No RNA template degradation > Higher cDNA yield
Improved sensitivity
Reduced 5’ / 3’ bias due to mispriming
Increased reliability
Greater percentage of full-length cDNA
complete sequence representation
Improved thermostability
high temperature cDNA synthesis
relaxes secondary structure
improved primer specificity
Consistent cDNA synthesis efficiency
wide template diversity
wide range of template amount
RNAse H reduced Thermostable ReverseTranscriptase: SuperScript III ™ RT
Good Reverse transcriptase is essential!

15. SuperScript™ III Platinum® One-Step RT-PCR

16. Which Polymerase?

17. Platinum® Taq DNA Polymerase
Which Taq polymerase?
Hot Start
- Increased specificity
- Reduced artifacts (less Primer Dimers)
Antibody Hot Start
- Fast activation
- Maximum activity in early cycles
Platinum® Taq DNA Polymerase
Hot Start Taq is a must!

18. PLATINUM® Taq automatic hot start
Initial Template
Denaturation
PCR Assembly
Temperature Cycling
94oC, 30s - 3 min
Inactive
Taq DNA Polymerase
Fully Active
Taq DNA Polymerase

19. UDG or no UDG?

20. Not all UDG kits are built equal!
Carry-over protection/comparison
Mix with UDG and dUTP
DNA
Amplicon with dUTP
Amplicon with dUTP X
20 cycles,
~1,000,000 fold difference
Not all UDG kits are built equal!

21. Which Detection system?

22. Detection chemistry? Decision, Decision… dsDNA specific stains
Ethidium bromide (used in first experiments)
SYBR Green I (most widely used today)
Probe-Based Systems (unlabeled primers plus probes)
TaqMan and TaqMan MGB probe system (5’ nuclease assay)
Dual Probe System, uses FRET between probes hybridized side by side to the amplicon
Molecular Beacon probe (hairpin probe, quencher and fluorophore are separated when hybridized to the amplicon)
Epoch probe (second structure probe, quencher and fluorophore are separated when hybridized to the amplicon)
Primer/oligo labeled systems
Amplifluor: hairpin primer with fluorophore and quencher
LUX™: primer with one fluorophore and no quencher, proprietary to Invitrogen
Decision, Decision…

23. Detection chemistry? SYBR Green I® TaqMan® Amplifluor™ LUX™
Quantiprobe®
Amplifluor™ Q
LUX™

24. Which detection system?
Monoplexing
Cost saving
Fast initial screening
Sybr Green I®
- Multiplexing
- High Specificity
- High Sensitivity
Probe based or
LUX™ primers
A detection system for every applications!

25. Light Upon eXtension! LUXTM (Light Upon eXtension) LUX™
The fluorescent intensity is modulated due to the proximity of the fluorophore to specific primary & secondary structures of the oligonucleotide. The design rules have been incorporated into the LUX Designer™. The labeled primer exhibits low fluorescence but incorporation during PCR produces a large increase in fluorescence
Light Upon eXtension!

26. Quenching effect
The fluorogenic LUX primer has an attached fluorophore at the 3’end and a short sequence tail on the 5’ end that is complementary to the 3’end of the primer. The resulting hairpin secondary structure provides optimal quenching of the attached fluorophore.
The quenching of fluorescence in duplex (ds DNA) is provided by the terminal dG-dC or dC-dG base pair when the dye is attached to a thymidine / cytosine within three nucleotides from the 3΄-end.

27. Competitive Audit Data
Certified LUX primers were compared to TaqMan® Gene Expression Assays from ABI and QuantiTect Gene Expression Assays from QIAGEN using Platinum Super-Mix UDG (1X ROX).
Standard curve generated from ten-fold serial dilutions of ORF clone (107 –102 copies).
ABI 7700
PCR Efficiency: 93%
R2:0.998
PCR Efficiency: 92%
R2:0.999
Quantitect IL6
PCR Efficiency: 94%
R2:0.996
LUX IL6
TaqMan IL6

28. Comparison of LUX™ to TaqMan
LUX™ sensitivity advantages

29. LUX™ Built-in control feature!
Melting curve analysis
Method:
Real-time PCR
After amplification: 60°C to 95°C (slow ramp)
Fluorescence signal is recorded continuously during the slow temperature ramp
Melting curve : Fluorescence signal vs. Temperature
Melting curve is converted to melting peaks by plotting –dF/dT vs Temperature
LUX™ Built-in control feature!

30. Advantages LUX™ versus TaqMan®
Specificity – Melting Curve
Fast control of the PCR product specificity without:
opening the tube and
running a gel
Provides accurate real time assessment of quality:
- eliminates risk of false positives
reduces risk of contamination

31. Advantages LUX™ versus TaqMan®
Alexa 546
TET
FAM
All detectors
Excitation
(nm)
Emission
FAM
492
517
JOE
520
548
TET
521
536
HEX
533
550
Alexa Fluor 546
554
570
Data from Molecular Probes spectra
Triplex amplification using standard protocol:
- 10,000 copies of Gene X are amplified using a LUX primer set labeled with Alexa Fluor 546
- 1,000 copies of Gene Z are amplified using a LUX primer set labeled with TET
- 100 copies of Gene Y are amplified using a LUX primer set labeled with FAM
Easy multiplexing!

32. Advantages LUX™ versus TaqMan®
Simple Primer Design with the new D-LUX™ Designer

33. 3 Choices to access LUX™ primers
Scientific project
D-LUX™ Designer
Self Service
EvoQuest™ LUX™
Custom Service
Certified LUX™ primers
- HSKG
Virus /Bacteria
Human genes
Flexibility!

34. Other convenient formats?

35. From cells to cDNA in ONE TUBE!
Cells Direct kit
Cell Lysis
No RNA isolation step required.
DNase treatment eliminates genomic DNA so you can be confident that results are due to cDNA amplification.
DNase Treatment
All done in 1 tube!
The cDNA synthesis is performed by SuperScript™ III RNase H- RT.
cDNA Synthesis
Application:
qPCR
From cells to cDNA in ONE TUBE!

36. From 1 cell to 10,000 cells without NAP!
Cells Direct kit
From 1 cell to 10,000 cells without NAP!

37. Room Temperature Stable
RTS kits
Optimized PCR SuperMix
Vialing
Proprietary Polymer Mix
Temperature-controlled Lyophilization
Packaging
Shelf-life = 1 year
Complete Cycle within 3 days
Room Temperature Stable

38. RTS and regular mixes have equal performances
RTS kits
Standard Curve RTS one step
y = (x) R 2
= 0.998 10 15 20 25 30 35 40
1.00E+00
1.00E+01
1.00E+02
1.00E+03
1.00E+04
1.00E+05
1.00E+06
Starting Concentration
Cycle Threshold
Standard Curve aqueous (SuperScript III one step)
y = (x) R 2
= 0.999 10 15 20 25 30 35 40
1.00E+00
1.00E+01
1.00E+02
1.00E+03
1.00E+04
1.00E+05
1.00E+06
Starting Concentration
Cycle Threshold
RTS and regular mixes have equal performances

39. What mix for what instrument?

40. Invitrogen qPCR Reagent Selection Guide
SYBR Green Detection
Fluorescent Probes/Primers
(LUX™, TaqMan®, etc.)
ABI 7000, 7700, 7900
Roche LightCycler
Any Instrument
DNA or cDNA
Platinum SYBR Green qPCR SuperMix-UDG w/ROX
Platinum SYBR Green qPCR SuperMix-UDG w/BSA
Platinum SYBR Green qPCR SuperMix-UDG
Platinum Quantitative PCR SuperMix-UDG w/ROX
Platinum Quantitative PCR SuperMix-UDG
RNA, 1-Step
SuperScript III Platinum SYBR Green One-Step qRT-PCR Kit w/ROX
SuperScript III Platinum SYBR Green One-Step qRT-PCR Kit w/BSA
SuperScript III Platinum SYBR Green One-Step qRT-PCR Kit
SuperScript III Platinum One-Step qRT-PCR Kit w/ROX
SuperScript III Platinum One-Step qRT-PCR Kit
RNA, 2-Step
SuperScript III Platinum SYBR Green Two-Step qRT-PCR Kit w/ROX
SuperScript III Platinum SYBR Green Two-Step qRT-PCR Kit w/BSA
SuperScript III Platinum SYBR Green Two-Step qRT-PCR Kit
SuperScript III Platinum Two-Step qRT-PCR Kit w/ROX
SuperScript III Platinum Two-Step qRT-PCR Kit
We will test AM’s on the most suitable kit for the two customer examples outlined earlier.
A solution for each problem!

41. SYBR Green LC qPCR versus Roche qPCR
Title of slide is set as Arial, Bold, Italic, size 24
Takeaway Box is also Arial, bold, italic, size 24– type the information first and then draw a gray 2 1/4 pt box around the copy block.
All text in Arial
Only italicize Titles and Text Boxes – use Title Case
Don’t do all caps for titles or column headings …use Title Case
No gray fill in objects where you have black type ... doesn’t copy well
No shadowing … on anything
Keep graphics very simple … 2D vs. 3D … keep colors compatible throughout presentation
Keep text within the boundaries so can be viewed well when on a large screen
HPRT primers were used with plasmid standards from 1x107 to 1x101 copies. Invitrogen SYBR Green LC (blue) quantified all 7 logs with a slope of –3.644 and an R-value of No template controls (NTC’s) were negative with the SYBR LC kit.
Roche’s FastStart DNA Master Plus (green) quantified 5 of 7 logs with a slope of –3.700 and an R-value of The NTCs along with the last two dilutions showed contamination.

42. What method of quantitation?

43. What Method and When

44. Absolute quantitation

45. Relative Quantitation: ΔΔCt Method
CtGOIs – Ctnorms = ΔCtSample
CtGOIc – Ctnormc = ΔCtCalibrator
ΔCtSample – ΔCtCalibrator = ΔΔCt
Fold Induction = 2- ΔΔCt
GOI = Gene of Interest
Norm = Normalizer (Housekeeping) gene

46. Where to find more info?

47. Invitrogen qPCR Central

48. Invitrogen qPCR Central

49. Invitrogen multidisplinary qPCR group
R&D qPCR group in Carlsbad, CA
Enzymologists in Carlsbad, CA
Chemists from Molecular Probes in Eugene, OR
Custom Primer manufacturing facility in Frederick, MA
EvoQuest service group in Carlsbad, CA
And more…

50. The end