1. بسم الله الرحمن الرحیم

2. بخش مهندسی بیوتکنولوژی کشاورزی
آموزش مبانی
Real time PCR
بخش مهندسی بیوتکنولوژی کشاورزی

3. History
The routine study of DNA became practical with the invention of the polymerase chain reaction (PCR) by Kary Mullis in 1983.
Despite advances in PCR, quantitation of DNA or RNA in cells remained a difficult task until 1993 when Russell Higuchi and collegues introduced real-time.
Higuchi R, Fockler C, Dollinger G, Watson R (1993) Kinetic PCR analysis: Real-time
monitoring of DNA amplification reactions. Biotechnology 11(9): 1026–1030.

4. Real Time PCR Application
Gene expression
Microarray and RNA seq validation
DNA methylation(QMSP, MSRE-PCR, MS-HRM )
SNP Genotyping & Alleilic discrimination
Copy Number Detection(CNV)
Pathogen detection and quantification

5. What exactly is real-time quantitative PCR?
Real-time PCR is the continuous collection of fluorescent signal from one or more polymerase chain reactions over a range of cycles.
Quantitative real-time PCR is the conversion of the fluorescent signals from each reaction into a numerical value for each sample.

6. Real-time PCR Principles
based on the detection and quantitation of
a fluorescent reporter
In stead of measuring the endpoint we focus on the first significant increase in the amount of PCR product.
The time of the increase correlates inversely to the initial amount of DNA template

7. Real-time PCR advantages
* not influenced by non-specific amplification * amplification can be monitored real-time * no post-PCR processing of products *~ high throughput * Broad dynamic range * requirement less RNA than conventional assays * most specific, sensitive and reproducible

8. Real-time PCR vs End Point Detection

9. Polymerase Chain Reaction (PCR)
PCR reaction components:
Tempelate
Primers
dNTP mix
MgCl2
Thermostable DNA polymerase
Buffer

10. Quantification in PCR
To understand real-time PCR, let’s imagine ourselves in a PCR reaction tube at cycle number 25…
What’s in our tube, at cycle number 25?
A soup of nucleotides, primers, template, amplicons, enzyme, etc.
1,000,000 copies of the amplicon right now.

11. Quantification in PCR What was it like last cycle, 24?
Almost exactly the same, except there were only 500,000 copies of the amplicon.
And the cycle before that, 23?
Almost the same, but only 250,000 copies of the amplicon.
And what about cycle 22?
Not a whole lot different. 125,000 copies of the amplicon.

12. Quantification in PCR ?
So, right now we’re at cycle 25 in a soup with 1,000,000 copies of the target.
What’s it going to be like after the next cycle, in cycle 26?

13. Quantification in PCR
Realistically, at the chain reaction progresses, it gets exponentially harder to find primers, and nucleotides. And the polymerase is wearing out.
So exponential growth does not go on forever!

14. Quantification in PCR
Let’s imagine that you start with four times as much DNA as I do…picture our two tubes at cycle 25 and work backwards a few cycles.
Cycle 25
Cycle Me
You 23
250,000
1,000,000 24
500,000
2,000,000 25
4,000,000

15. Quantification in PCR
So, if YOU started with FOUR times as much DNA template as I did… …Then you’d reach 1,000,000 copies exactly TWO cycles earlier than I would!

16. Quantification in PCR What if YOU started with EIGHT times
LESS DNA template than I did?
Cycle Me
You 25
1,000,000
125,000 26
2,000,000
250,000 27
4,000,000
500,000 28
8,000,000
Cycle 25

17. Quantification in PCR
What if YOU started with EIGHT times LESS DNA template than I did?
You’d only have 125,000 copies right now at cycle 25…
And you’d reach 1,000,000 copies exactly THREE cycles later than I would!

18. Amplification plot

19. Amplification plot

20. Amplification plot

21. Exponential phase
Linear ~20 to ~1500

22. Exponential phase
Linear ~20 to ~1500

23. Threshold Cycle (CT) 21 23 26

24. Serial Dilution
SERIES OF 10-FOLD DILUTIONS

25. Efficiency calculation
threshold Ct

26. Efficiency calculation

27. Optical Detection Systems
2a. excitation filters
2b. emission filters
1. halogen tungsten lamp
4. sample plate
3. intensifier
5. ccd detector 350,000 pixels
Chapter1 from rna methodology book

28. Fluorescence Chemistry
DNA binding agents
SYBR Green, Eva Green, BOXTO, LCGreen®, SYTO® 9 and BEBO
Hydrolysis probes
TaqMan
hybridization probes
FRET

29. Fluorescence resonance energy transfer
FRET is a mechanism describing energy transfer between two light-sensitive molecules (chromophores).
A donor chromophore, initially in its electronic excited state, may transfer energy to an acceptor chromophore through nonradiative dipole–dipole coupling. The efficiency of this energy transfer is inversely proportional to the sixth power of the distance between donor and acceptor, making FRET extremely sensitive to small changes in distance
FRET depends on the donor and acceptor molecules being in close proximity (10– 100 A)

30. DNA binding agents SYBR® Green I advantages Low cost assay
Easy design and set up
Useful for generating melt curves and HRM analysis
SYBR® Green I disadvantages
Non specific system
Not adapted to multiplex

31. Apply Excitation Wavelength5’ 3’ 5’ 3’ 5’ 3’ 5’ 3’
Extension
Taq ID ID 5’ 3’ 5’ 5’ ID ID ID
Taq 5’ 3’
Apply Excitation
Wavelength l
Taq ID 5’ 3’ ID ID 5’ 5’ ID ID
Taq 3’ l

32. Hydrolysis probes Double-Dye probes advantages
Widely used, several modifications possible
Multiplex capabilities
Double-Dye probes disadvantages
More expensive than SYBR® Green I
Problem in Design

33. TaqMan® probe types
TaqMan probe
TaqMan MGB probe

34. Molecular Beacon

35. hybridization probes

37. Amplification Plots & Basic Terms
Rn: Fluorescence signal normalized with passive reference dye signal (Fluorescence/dye signal)
Passive Reference Dye: An internal reference to correct well to well non‐PCR fluctuations. ROX is the most commonly used reference dye.
ΔRn: Fluorescence signal with baseline subtracted (background signal subtracted)
Baseline: background noise level before a significant amplification occurs (3‐15 cycles)

38. Assay Validation Specificity Analysis Precision and Variability
Melt Curves
Amplicon Size Analysis
Sequencing
Precision and Variability

39. The hallmarks of an optimized qPCR assay
Efficiency:amount of PCR product dubles during each cycle of exponential amplification
Specificity:target DNA is the only reaction product
Sensetivity:broad linear range that encompases the entire range of templet concentration
Reproducibility:variability across assay replicates is minimal

40. Determining Gene Expression Changes

41. Why study RNA?
All cell and tissue functions are ultimately governed by gene expression
Fundamental themes in RNA studies:
Abundance
Structural
Cell-free in vitro translation
Synthesis of cDNA
Chapter1 from rna methodology book

42. Types of RNA rRNA(most abundant) mRNA(most diverse) tRNA
Different class of Small RNAs
Chapter1 from rna methodology book

43. Gene expression methods
Northern blot
Nuclease protection
SAGE
SSH
cDNA AFLP
Micro array
Semi quantative PCR
Real time PCR
Digital PCR
RNA-seq

44. Pipeline for gene expression analysis by Real time PCR
RNA extraction
RNA qualification and quantification
cDNA synthesis
PCR reaction

45. RNA qualification and quantification
UV spectrophotometry
Microfluidic analysis (2100 Bioanalyzer, Experion,qiaexcel )
RNA binding dyes(RiboGreen)
Qualification
Agarose Gel

46. cDNA synthesis

47. cDNA synthesis
There are three ways to prime a reverse transcriptase reaction:
Oligo-dT
Random primers
Assay-specific primers

48. DNA Polymerase
The heart of the modern PCR is the addition of a thermostable DNA polymerase
Hot start by:
Antibody-mediated(1min 94ºC)
Chemical-mediated (10min 94ºC)
Organism
Thermus aquaticus MW
94KDa
Number of amino acids
832
Single chain or subunits
single
Extension rate
2-4 Kb/min
Half 95ºC
40 min
Processivity
50-60 bases
5’–3’ exonuclease activity
yes

49. One-step and Two-step RT-PCR
Procedure
Advantages
Two-step RT-PCR
Multiple PCRs from a single RT reaction
Flexibility with RT primer choice
Enables long-term storage of cDNA
One-step RT-PCR
Easy handling
Fast procedure
High reproducibility
Low contamination risk

50. Effect of RT volume added to real time PCR

51. Primer and probe design
Length of PCR product should ideally be less than 250 bp
Avoid complementary sequences within and between primers and probes
Avoid mismatches
Avoid a 3'-end T as this has a greater tolerance of mismatch
Length: 18–28 nucleotides
GC content: 40–60%
The probe melting temperature in general should be ~10ºC higher than the forward or reverse primer.
Do not put G at the 5’ end of the probe as this will quench reporter fluorescence

52. Primer and probe design Tools
Primer3 ( bin/primer/primer3_www.cgi)
Idt(
Beacon designer
Primer Express® (
Premier Biosoft International (

53. Quantification strategies
Absolute quantification Standard curve method
Relative quantification
Standard curve method
With PCR efficiency correction
Comparative CT Method
Without PCR efficiency correction

54. Absolute quantification
The absolute quantitation assay is used to quantitate unknown samples by interpolating their quantity from a standard curve

55. Absolute quantification
Features of an appropriate standard
Primer and probe binding sites identical to the target to be quantified
Sequence between primer binding sites identical or highly similar to the target sequence
Sequences upstream and downstream from the amplified sequence identical or similar to the“natural” target
Equivalent amplification efficiencies of standard and target molecules

56. Absolute quantification

57. Housekeeping Genes
Genes that are widely expressed in abundance and are usually used as reference genes for normalization in real-time PCR with the assumption of ‘constant expression’.
The current trend is first to check which housekeeping genes are suitable for the target cell or tissue and then to use more than one of them in normalization.

58. Housekeeping Genes
A Reference Gene is aimed to normalize possible variations during:
Sample prep & handling (e.g use the same number of cells from a start)
RNA isolation (RNA quality and quantity)
Reverse transcription efficiency across samples/experiments
PCR reaction

59. Relative quantification by Standard curve method
dilutions target DNA
dilutions reference DNA
target
primers
reference
triplicates cDNA

60. Relative quantification by Standard curve method
‘copy number’ target gene control
Dilution curve target gene
‘copy number’ target gene experimental

61. Relative quantification by Standard curve method
‘copy number’ reference gene experimental
Dilution curve reference gene
‘copy number’ reference gene control

62. Relative quantification by Standard curve method

63. Relative quantification
Determination of the changes in steady state mRNA levels of a gene across multiple samples and expresses it relative to the levels of another RNA.
Calibrator: A single reference sample used as the basis for relative-fold increase in expression studies
To determine the level of expression, the differences (Δ) between the
threshold cycle (Ct) or crossing points (CP) are measured.
Comparative CT Method (ΔΔ CT Method)

64. Relative quantification
gene expression can be relative to:
an endogenous control, e.g. a constant expressed reference gene or another GOI
a reference gene index, e.g. consisting of multiple averaged endogenous controls

65. Without PCR efficiency correction

66. Without PCR efficiency correction
if the PCR efficiency is only 0.9 instead of 1.0, the resulting error at a threshold cycle of 25 will be 261%. The calculated expression level will be 3.6-fold less than the actual value.

67. The efficiency corrected calculation method

68. Example

69. Example ΔCt _GAPDH = Ct control - Ct treated ΔCt _GAPDH = 14.0 – 13.7
ΔCt _GAPDH = Ct control - Ct treated
ΔCt _GAPDH = 14.0 – 13.7
ΔCt _GAPDH = 0.3

70. Factors affecting the accuracy of Real-Time PCR
Pre-analytical steps
Tissue sampling and storage, RNA extraction and storage, RNA quantity and quality control
Optimized RT and PCR performance
specificity, sensitivity, reproducibility, and robustness
Post-PCR data procession
data acquisition, evaluation, calculation and statistics

71. Softwares
REST
Q-Gene
qBASE plus
DART-PCR
linReg PCR