1. Introduction to Real-Time PCR
Dr. Chaim Wachtel
April 11, 2013

2. Real-Time PCR What is it? How does it work
How do you properly perform an experiment
Analysis

3. The Nobel Prize in Chemistry 1993 was awarded "for contributions to the developments of methods within DNA-based chemistry" jointly with one half to Kary B. Mullis "for his invention of the polymerase chain reaction (PCR) method"and with one half to Michael Smith "for his fundamental contributions to the establishment of oligonucleotide-based, site-directed mutagenesis and its development for protein studies".
Michael Smith

4. PCR – A simple idea Polymerase Chain Reaction: Kary Mullis (1983)
In vitro method for enzymatically synthesizing DNA
The reaction uses two oligonucleotide primers that hybridize to opposite strands and flank the target DNA sequence that is to be amplified
A repetitive series of cycles gives exponential accumulation of a specific DNA fragment
Template denaturation
Primer annealing
Extension of annealed primers by the polymerase
The number of target DNA copies doubles every PCR cycle (20 cycles  220≈106 copies of target)

5. Principle of PCR

6. Difference PCR vs real-time PCR?
Fluorescence is measured every cycle (signal  amount of PCR product).
Curves rise after a number of cycles that is proportional to the initial amount of DNA template.
Comparison with standard curve gives quantification.

7. Real-Time and End Point

8. MIQE: the minimum information for the publication of qPCR experiments.

12. The mRNA of the Arabidopsis Gene FT Moves from Leaf to Shoot Apex and Induces Flowering
Tao Huang, Henrik Böhlenius, Sven Eriksson, François Parcy, and Ove Nilsson
Science 9 September 2005:
2005: Signaling Breakthroughs of the Year

13. Retraction
WE WISH TO RETRACT OUR RESEARCH ARTICLE “THE MRNA OF THE ARABIDOPSIS GENE FT MOVES
from leaf to shoot apex and induces flowering” (1). After the first author (T.H.) left the Umeå Plant Science Centre for another position, analysis of his original data revealed several anomalies.
It is apparent from these files that data from the real-time RT-PCR were analyzed incorrectly.
Certain data points were removed, while other data points were given increased weight in
the statistical analysis.
When all the primary real-time RT-PCR data are subjected to correct statistical analysis, most of the reported significant differences between time points disappear.
Because of this, we are retracting the paper in its entirety.

14. Real-Time Machines How do they work What can you do with one
Gene expression
SNP detection
DNA detection (quantify)
How do you use them
Experiment design
Everything you need to know and more about RNA and RT-PCR

15. First real-time PCR, 1991 PCR tube in thermocycler
spectrofluorometer
fiberoptic
“Fifty Years of Molecular Diagnostics”
Clin Chem Mar;51(3): (C.Wittwer, ed.)

16. First commercial real-time PCR instruments
ABI 7700 – laser/fiberoptic-based
ABI 5700 – CCD camera-based
Idaho Technology LightCycler – capillary tubes

17. RT-PCR machines at Bar Ilan
7900HT Fast Real-Time PCR System
(Sol Efroni’s lab)
AB StepOnePlus
Fast Real-Time PCR System
Qiagen’s Rotor-gene
(Oren Levy’s lab)
Thermo PikoReal
(Bachelet Lab)
Bio-Rad CFX-96

18. Rotor-gene

19. Probing alternatives Non-specific detection
Dyes: SYBR Green I, BEBO, BOXTO, EvaGreen...
Specific detection
TaqMan probe
Molecular Beacon
Light-Up probe
Hybridization probes
Primer based detection
Scorpion primers
QZyme
Lux primers

20. SYBR Green binds to dsDNA
SYBR Green binds to DNA, particularly to double-stranded DNA, giving strongly enhanced fluorescence.
SYBR Green is sequence-dependent!

21. The TaqMan Probe
The TaqMan probe binds to ssDNA at a combined annealing and elongation step.
It is degraded by the polymerase, which releases the dye from the quencher.

22. Multiplex Q-PCR
Detection of two (or more) different target sequences in the same reaction.

23. qPCR technical workflow
DNA
Extraction
Data
Analysis
Sampling
qPCR
RNA
Extraction
DNase
treatment
Reverse
Transcription

24. Nucleic acid isolation and purification24

25. Overview Sampling Accessibility and lysis Commonly used techniques
RNA considerations
Quality control
The process of purifying nucleic acids is often taken for granted
Researchers frequently underestimate the importance of choosing a method that gives them not only good recoveries but also molecular biology-grade samples
A lot of time and money are invested in molecular biology applications, so its important to isolate highly pure nucleic acids to begin with
Without an effective method for nucleic acid purification, the quality of the results will be compromised. 25

26. Why sample preparation?
Make target available
Remove inhibitors
Remove fluorescent contaminants
Preserve target integrity
Concentrate target

27. Path Reverse Transcription Real-time PCR DNA RNA Purification
Isolation
Disruption
mRNA
Genomic DNA
Total RNA
Plasmid DNA
Fragment DNA
Nuclear RNA
Phage DNA

28. Accessibility Sample disruption and homogenization Mechanical
Grinding, Sonication, Vortexing, Polytron
Physical
Freezing
Enzymatic
Proteinase K, Lysozyme, Collagenase
Chemical
Guanidinium isothiocyanate (GITC), Alkali treatment, CTAB
Disruption is the physical breakdown of cellular components (walls and plasma membranes) using mechanical and/or enzymatic techniques. Successful disruption will release all RNA contained in the sample and produce high yield
Homogenization will reduce the viscosity of the cell lysate and incorporate large particles formed in the disruption step into a homogenous mixture. Successful homogenization will reduce column clogging and generate high yield
Nämn även beta-mercaptoetanol
hexadecyltrimethylammonium bromide (CTAB)
Mechanical methods for disrupting fresh tissue and cells include homogenization with a Dounce or with a mechanical homogenizer (such as the Brinkmann Polytron¥), vortexing, sonication, French press, bead milling, and even grinding in a coffee grinder! 28

29. Lysis Complete or partial lysis? Chaotropic lysis buffers:
SDS, GITC, LiCl, phenol, sarcosyl
Gentle lysis buffers:
NP-40, Triton X-100, Tween, DTT
More strong: urea, chloroform,
They are chaotropic detergents
Weak lysis could used to du plasma membrane specific lysis
NP-40 = Igepal CA-630
Sarcosyl is also called N-laurylsarcosine and is mainly used for plant samples 29

30. Purification principles
Characteristics of nucleic acids
Long, unbranched, negatively charged polymers
Examples:
Differential solubility
Precipitation
Strong affinity to surface
Factors:
pH, [salt], hydrophobicity

31. Purification techniques
Solution based- eg Tri reagent, CsCl gradient
Precipitation- ethanol, needs salt, multiple factors can influence precipitation
Membrane based- spin columns (Qiagen and the like)
Magnetic bead based
Typical protocol:
Stabilisation of the sample
Lysis and homogenisation in denaturing buffer / beta-mercaptoethanol
Addition of ethanol for adjustment of binding conditions
Selective RNA binding (nature and concentration of the chaotrope)
Stringent salt wash – removal of proteins, carbohydrates and most gDNA
DNAse I digest (optional)
Second stringent wash
Ethanolic wash
Dry spin / extended vacuum step
Elution in TE buffer or RNase-free water 31

32. Solution based isolation
Most methods use hazardous reagents
Phenol/Chloroform extraction
Proteins, lipids, polysaccharides go into the organic phase or in the interphase.
DNA/RNA remains in aqueous phase
Caesium chloride density gradient ultracentrifugation
Time consuming
Acid guanidine phenol chloroform extraction
Commonly called TRIzol

33. Precipitation purification
Nucleic acids precipitate in alcohols
Salt (NaCl, NaAc) facilitates the process
Important factors: Temperature, time, pH, and amount

34. Membrane based isolation
Anion exchange technology
Spin column / silica gel membrane
Chaotropic salts (e.g. NaI or guanidine hydrochloride) bind H2O molecules
Loss of water from DNA changes shape and charge
DNA binds reversibly to silica membrane

35. Purification – GITC vs. column
Organic liquids
Pro:
Higher yield
Can handle larger amounts of cells
Better for troublesome tissues (fatty tissue, bone etc)
Con:
Higher DNA contamination (for RNA isolation)
Separate DNase I digestion with additional purification
Spin columns
Pro:
Less contaminating DNA (for RNA isolation)
On column DNase digestion Less loss of RNA
Higher quality
Easy to use
Con:
Limited loading capacity
More expensive (?)

36. RNA Considerations
RNA is chemically and biologically less stable than DNA
Extrinsic and intrinsic ribonucleases (RNases)
Specific and Nonspecific inhibitors
Speciellt känsligt är RNA vid >65 grader och i basisk miljö
RNase finns överallt, även inne i alla celler
Successful isolation depends on the inactivation of ribonucleases which degrade RNA rendering it useless for downstream analysis
Reagents and plasticware should either be guaranteed RNase-free by the supplier or treated with DEPC
Inactivated by the GITC and beta-mercaptoethanol present in the lysis buffer 36

37. Stabilizing conditions
Work on ice
Process immediately
Flash freeze sample in liquid nitrogen and store at
-70°C until later use
Store samples in stabilization buffer
RNAlater:
Permeates the sample and freezes the gene expression profile
Entirely non-hazardous
Can be used at ambient temperature
Sample can be repeatedly frozen and thawed without reduction in RNA quality 37

38. Storage of nucleic acids
Nuclease-free plasticware
Eluted in nuclease-free water, TE or sodium citrate solution
RNA:
Neutral pH to avoid degradation
Aliquot sample to avoid multiple freeze-thaw cycles
Isolated RNA should be stored at -20 deg C or -70 deg C for even better protection in ethanol and not water.
milli Q / double distilled lab water is often at acid pH 38

39. Quality Control Spectroscopic methods
Concentration, [NA] = A260 x e mg/ml
Purity: A260 / A280 (≈1.8 for DNA, 2.0 for RNA)
Dyes
Quantification by fluorescence of DNA/RNA-binding dyes (Qubit)
Electrophoresis (28S and 18S bands)
Kanke ändra bort epsilon till faktor.
Extinction coefficient for DNA is 50, RNA it’s 40. Assuming l = 1 cm.
Pure DNA has ratio of , pure RNA has
Optimise the quantity of staring material
Ensure reproducibility by always weighing the sample
Thereby avoid exceeding the lysis capacity of the buffer
Check lysis buffer for precipitation
If following a vacuum protocol, then set the strength of the vacuum with empty wells
If following a spin protocol, then use an appropriate g force at every spin step
Determine the yield and quality spectrophotometrically or using the Agilent 2100 Bioanalyser 39

40. What is the BioAnalizer?
Microfluidic separations technology
RNA - DNA - Protein
1µl of RNA sample (100 pg to 500 ng)
12 samples analyzed in 30 min
Integrated analysis software:
Quantitation
Integrity of RNA

41. Bioanalyzer

45. RNA Integrity: RQI Good RNA Quality Bad RNA Quality
10 RNA Quality Indicator 1

46. Publications on RNA integrity

47. DNase I treatment of RNA samples
RT, No DNase
No RT, No DNase
RT, DNase
No RT, DNase

48. qPCR technical workflow
DNA
Extraction
Data
Analysis
Sampling
qPCR
RNA
Extraction
DNase
treatment
Reverse
Transcription

49. Reverse transcription RT49

50. Outline Priming efficiency Reproducibility
Properties of Reverse transcriptase
RNA concentrations

51. General description of RT reaction
Reverse Transcriptases are RNA-dependent* DNA polymerases that catalyze first strand DNA synthesis in presence of a suitable primer+ as long as it has a free 3’ OH end.
*Can use also single strand DNA as template.
+ Can be either RNA or DNA.

52. RT priming

53. RT with Gene-Specific Priming

54. RT with Oligo(dT) Priming

55. RT with Random Hexamer Priming

56. Real-time PCR using different RT primers

57. Real-time PCR with different RT primers

58. Dependence on priming strategy

59. Dependance of priming method
Gene
b-tubulin
CaVID
GAPDH
Insulin II
Glut 2
hexamers
19,5
26,5
15,8
16,9
27,5
oligo dT
18,1
28,8
16,6
15,9
28,4
GSP
18,8
28,7
16,4
17,4
31,8
mix
19,1
27,9
16,3
29,3
max DCt
1,4
2,3
0,8
1,5
4,4
RT priming method

60. Specificity of specific priming
PCR primers used
b-tubulin
CaVID
GAPDH
Insulin II
Glut 2
18,8
28,7 19
30,6 27
18,7
19,9
22,8 -
23,4
30,1
16,4
20,1
29,7
23,5
31,6 20
17,4 31
25,8
31,9
22,7
31,8
no RT primer
27,6
33,7
23,6
23,1
32,6
RT primers used
NTC ~ 35

61. 60ºC 37ºC Algorithm: mfold GAPDH 3’ 24 unpaired bases

62. Comparison of reverse transcriptases
Temp
MMLV RNase H- Minus (Promega, Germany) 37
M-MLV (Promega)
Avian Myeloblastosis Virus (AMV) (Promega) 37
Improm-II (Promega)
Omniscript (Qiagen, Germany) 37
cloned AMV (cAMV) (Invitrogen, Germany) 45
ThermoScript RNase H- (Invitrogen) 50
SuperScript III RNase H- (Invitrogen) 50
Ref: Ståhlberg et al. Comparison of reverse transcriptases in gene expression analysis. Clin.Chem. 50(9); (2004)

63. 100 – fold variation in RT yield

64. 8 transcriptases tested on 6 genes

65. Experimental design to study linearity

66. Effect of carrier
- tRNA
+ tRNA

67. Effect of carrier
- tRNA
+ tRNA

68. RNA dilutions Oligo(dT) Random Hexamers Water Yeast tRNA
Fundera på att ha på flera sen sammanfattande. 68

69. Conclusions
The RT reaction shows higher technical variability than QPCR
There is no optimum priming strategy
Gene specific primers must target accessible regions
The RT yield changes over 100-fold with the choice of reverse transcriptase
The yield variation is gene specific
RT yield is proportional to the amount of template in presence of proper carrier
Typical RT yield is %
RT-QPCR is highly reproducible as long as the same protocol and reaction conditions are used
The efficiency of the RT reaction varies from gene to gene and depends on the conditions – run the RT of all samples using exactly the same protocol and reagents under the same conditions