The principle of SYBR real-time PCR is a standard PCR reaction carried out in the presence of a dye, SYBR, which fluorescence when intercalated in the DNA helix. - PowerPoint PPT Presentation

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The principle of SYBR real-time PCR is a standard PCR reaction carried out in the presence of a dye, SYBR, which fluorescence when intercalated in the DNA helix.

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Quantification of mRNA using real-time PCR Abdelilah Soussi Gounni, Ph.D Associate Professor Manitoba Research Chair Department of Immunology University of Manitoba – PowerPoint PPT presentation

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Title: The principle of SYBR real-time PCR is a standard PCR reaction carried out in the presence of a dye, SYBR, which fluorescence when intercalated in the DNA helix.


1
Quantification of mRNA using real-time PCR
Abdelilah Soussi Gounni, Ph.D Associate
Professor Manitoba Research Chair Department of
Immunology University of Manitoba
2
  • Objectives
  • Introduction to qPCR
  • Chemistries used in qPCR
  • Different platforms pro and con
  • Primer design and probe design
  • RT Methods of priming.
  • Sample preparation, RNA extraction.
  • Quantification strategies
  • QPCR application

3
Why Measure Gene Expression?
  • More abundant genes/transcripts are more
    important.
  • Each cell has a standard expression
    profile/signature.
  • Assumption that gene expression levels correspond
    to protein levels.

4
Why Measure Gene Expression?
  • Gene expression profiles represent a snapshot of
    cellular metabolism or activity at the molecular
    scale
  • Gene expression profiles represent the cumulative
    interactions of many intracellular events or
    phenomena

5
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6
Real time PCR Generalities
  • The real-time polymerase chain reaction uses
    ?uorescent reporter dyes to combine DNA
    ampli?cation and detection steps in a single tube
    format.
  • Increase in ?uorescent signal is recorded during
    the assay.
  • Fluorescent signal is proportional to the amount
    of DNA synthesized during each amplification
    cycle.

7
Real time PCR Generalities
  • Individual reactions are characterized by the
    cycle fraction at which ?uorescence ?rst rises
    above a de?ned  background ?uorescence, a
    parameter known as the threshold cycle (Ct) or
    crossing point (Cp).
  • Consequently, the lower the Ct, the more abundant
    the initial target. 
  • The homogeneous format eliminates the need for
    post-ampli?cation manipulation and signi?cantly
    reduces hands-on time and the risk of
    contamination.

8
A hypothetical amplification plot
  • The amplification plot is the plot of
    fluorescence signal vs PCR cycle number.
  • The baseline is defined as the PCR cycles in
    which a signal is accumulating but is beneath the
    limits of detection of the instrument.

9
How the signal of Real time PCR is quantified
  1. The signal measured during these PCR cycles is
    used to plot the threshold.
  2. The threshold is calculated as 10 times the
    standard deviation of the average signal of the
    baseline fluorescent signal.
  3. A fluorescent signal that is detected above the
    threshold is considered a real signal that can be
    used to define the threshold cycle (Ct) for a
    sample.
  4. The Ct is defined as the fractional PCR cycle
    number at which the fluorescent signal is greater
    than the minimal detection level.

10
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12
Three main chemistries are used in Real time PCR
  • 1- Intercalating dyes
  • SYBR-Green this dye fluoresce upon light
    excitation when bound to double stranded DNA.
  • Advantage
  • Cost effective.
  • Easily added to assays
  • Amplification products can be verified by the
    use of melting curves.
  • one Ct or so more sensitive than probe-based
    assays.
  • Disadvantage
  • Lack of specificity and fluorescence varies with
    amplicon length.

Adapted from Bustin S.
http//www.scitopics.com/Real_time_Polymerase_Chai
n_reaction.html
13
Three main chemistries are used in Real time PCR
  • 1- Intercalating dyes
  • SYBR-Green

14
Three main chemistries are used in Real time PCR
  • 1- Intercalating dyes
  • SYBR-Green

15
Three main chemistries are used in Real time PCR
  • 2-Fluorophores attached to primers
  • Invitrogen's Lux or Promega's Plexor primers.
  • Advantage Relatively inexpensive and
    amplification products can be verified by melting
    curves.
  • Specificity depends on the primers.
  • Usually company-specific design software needs
    to be used for optimal performance.

16
LUX (light Upon eXtension)
  • D-LUX detection technology uses one primer
    labeled with a single fluorophore and a
    corresponding unlabeled primer.
  • Both custom-synthesized according to the target
    of interest. Typically 20-30 bases in length,
    LUX Primers are designed with a fluorophore near
    the 3end in a hairpin structure.
  • This configuration intrinsically renders
    fluorescence quenching capability, making a
    separate quenching moiety unnecessary.
  • Once the primer becomes incorporated into the
    double-stranded PCR product, the fluorophore is
    de- quenched.

17
LUX (light Upon eXtension) Primers
Invitrogen Web site
18
Three main chemistries are used in Real time PCR
  • 3. Hybridisation-probe based methods TaqMan or
    Molecular Beacons.
  • Most specific, as products are only detected if
    the probes hybridize to the appropriate
    amplification products.
  • Many variations on this theme, with melt curve
    analysis possible for some chemistries (UBI).
  • Main disadvantages are cost, complexity and
    occasional fragility of probe synthesis.
  • There are potential problems associated with the
    fact that probe-based assays do not report primer
    dimers that can interfere with the efficiency of
    the amplification reaction.

19
TaqMan Probes how it works
  • Oligonucleotides that contain a fluorescent dye,
    typically on the 5' base, and a quenching  dye,
    typically located on the 3' base.
  • When irradiated, the excited fluorescent dye
    transfers energy to the nearby quenching dye
    molecule rather than fluorescing, resulting in a
    nonfluorescent substrate.
  • TaqMan probes are  designed to hybridize to an
    internal region of a PCR product.
  • During PCR, when the polymerase replicates a
     template on which a TaqMan probe is bound, the
    5' exonuclease activity of the polymerase cleaves
    the probe.
  •  This separates the fluorescent and quenching
    dyes and FRET no longer occurs. Fluorescence
    increases in each  cycle, proportional to the
    rate of probe cleavage. 

20
TaqMan probe
21
Platforms or Cyclers
  • Two very different principles for commercial
    thermocyclers block-and air based.
  • The block based cyclers
  • They worked by heating and cooling a metal
    block in which the samples are placed.
  • The heat is usually moved by Peltier elements
    which are semiconductors which move heat when a
    current is applied to the cells.
  • If the polarity of the current is reversed, the
    direction of the heat transport is also reversed.

22
Heating Block disadvantage
  • Heating blocks are relatively slow and changes
    typically the temperature 1 to a few degree
    celcius per second.
  • Heating blocks are difficult to heat uniformly
    across the plate.

23
Air cyclers
  • The temperature changes by means of heated air.
  • The samples are in 96-well plates, or in
    capillaries, the later conduct the heat very
    rapidly.
  • Traditionally, air cyclers can typically
    accommodate less samples than plate cyclers, but
    the time to complete a run is much shorter, due
    to the rapid heat conduction in the system.
  • Not anymore Roche new system.
  • The temperature gradient is typically 20 degrees
    C per second.

24
Designing primers
  • Look at public data bases first
  • Rt primer DB
  • htpp//medgen.ugent.be/rtprimer db
  • Primer bank htpp//pga.mgh.harvard.edu/primerbank/
    index. Html
  • Real-time primers set(http//www.realtimeprimers.o
    rg

25
Conti..
  • If you found your probe and primers
  • Then input the sequences into blast
    http//www.ncbi.nlm.nih.org
  • Examine the sequences for possible errors,
    polymorphisms and avoid these regions for primer
    or probe design.
  • Avoid direct repeat in the target sequences
    hybridization to alternative site in repetitive
    regions results in non productive binding of
    primers, a reduction in the efficiency of DNA
    amplification.
  • Sensitivity reduction of the assay

26
Conti...
  • -When possible use a primer sequence in
    boundaries between two exons separated by along
    introns no need for DNAase treatment due to
    genomic contamination.
  • -try to have a short amplicon as possible (60 to
    150 bp) with GC content of 60 or less to ensure
    efficient denaturation.
  • -Choose primer that target mille of your target
    gene

27
Isolation of RNA
  • RNA isolation based on 3 principals
  • Lysing of cells to release nucleic acids
  • Inactivation of RNases
  • Separation of RNA from DNA
  • Old School
  • Tissue Homogenization (Grinding on Dry Ice/LN2)
  • Hot Phenol/SDS or Guanidium thiocyanate
  • CsCl gradient centrifugation
  • New
  • Chaotropic Salt Isolation (Qiagen)
  • Trizol (Invitrogen)
  • Isolation of mRNA from total RNA (oligo dT)
  • Isolation of cytoplasmic RNA

28
RNA assessment
  • Many strategies are used
  • Spectrophotometer widely used
  • Riobogreen
  • Agilent bioanalyser
  • Nanodrop
  • Biorad experion
  • No method leads to the same data!!
  • Extremely important Use the same methods for
    measuring all samples
  • Do not compare data of samples analysed by
    different methods
  • Butin SA et al 2005

29
RNA quality
  • Widely used approaches
  • ratio A260/280 between 1.9 to 2.1 ( 100
    purity)
  • Gel analysis of ribosomal RNA intact 18S and
    28S (doubling intensity on gel of 28S compared
    to 18S).
  • Agilent bio-analyzer/ BioRad experion
    microfluidic
  • Nanodrop
  • Free of contamination
  • DNA
  • Proteins
  • Inhibitors carry over chemicals from
    purification steps
  • These contamination may depends on the tissue or
    cell from which RNA come from.

30
RNA quality
  • Best approach use 3- 5 assay using GAPDH as
    target gene.
  • Adopted by microarray users
  • Independent of ribosomal integrity
  • Accepted as conventional techniques applied to
    end point PCR assays
  • Auer et al, 2003

31
Primers and amplicon for inhibitors assessment
and 3-5assay
32
RNA quality assay
33
Effect of Primers concentration on QPCR
Nolan T et al, 2006
34
cDNA priming
  • Oligo dT
  • suppose that the RNA is intact.
  • non suitable if looking for
  • Splice variants
  • Sequences long 3 UTR
  • Non poly genes

35
cDNA priming
  • Random hexamers
  • not equal efficiencies of RT of all target
    genes.
  • Absence of linear correlation between input RNA
    and cDNA yield when a specific target is
    measured.
  • Gene specific primers
  • Good choice if the RNA is not limited.
  • Useful for extremely rare mRNA.

36
cDNA Priming
  • 15 nt Random primers tend to be the choice
    taking in account recent studies comparing all
    methods.
  • This method has been shown to yield 80 of the
    template compared to 40 with random hexamers
  • RT step tends to be the source of variability in
    PCR

37
  • Practical lab considerations

38
Steps involved in planning RT-QPCR
39
RT-QPCR experimental workflow
40
RT-qPCR
41
Cont...
42
Cont...
43
Conti
44
In summary
  • Prepare well all your samples ( start with a
    simple experiment first).
  • Be sure that you have all what you need.
  • Never use other people reagents.
  • Work in dedicated area for RT-QPCR.
  • Prepare all the controls.

45
Ct threshold cycle
  • The cycle at which the fluorescence exceeds a
    detection threshold, the Ct (threshold cycle)
    correlates to the number of target cDNA molecules
    present in the added cDNA.
  • Consequently, by comparison to a calibration
    curve, it is possible to quantify in absolute
    amounts the number of target molecules in added
    cDNA samples.

46
Relationship between standard curve and
amplification
47
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48
How the signal of Real time PCR is quantified
  1. The signal measured during these PCR cycles is
    used to plot the threshold.
  2. The threshold is calculated as 10 times the
    standard deviation of the average signal of the
    baseline fluorescent signal.
  3. A fluorescent signal that is detected above the
    threshold is considered a real signal that can be
    used to define the threshold cycle (Ct) for a
    sample.
  4. The Ct is defined as the fractional PCR cycle
    number at which the fluorescent signal is greater
    than the minimal detection level.

49
Comparative Ct method
  • The Ct values of different samples are used to
    calculate the relative abundance of template for
    each sample.
  • In this plot the solid line crosses the threshold
    at PCR cycle number 18 whereas the dotted line
    crosses at 20. By subtracting 18 from 20, there
    is a two-cycle difference between these two
    samples or a ?Ct of 2.
  • Due to the exponential nature of PCR the ?Ct is
    converted to a linear form by 2-(?Ct) or fourfold
    difference.
  • This calculation is used when performing a
    relative quantitation analytical method.

50
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51
Acceptable Standard curve
52
Unacceptable standard curve
53
Dissociation curves are useful for determining
the presence of multiple species in the sample
  • Every PCR product melts at a characteristic
    temperature, its Tm (melting temperature).
  • A characteristic melting peak at the amplicon's
    Tm will usually distinguish it from amplification
    artifacts that melt at lower temperatures in
    broader peaks.
  • In the PCR, these are typically primerdimer
    artifacts or non-specific amplicons.
  • For standard analysis, samples are first melted
    at 95 C, and then equilibrated at 60 C before
    being slowly re-heated (dissociated) back to 95
    C. The centre of each peak reflects Tm.

54
Dissociation curves are useful for determining
the presence of multiple species in the sample
55
Dissociation curves are useful for determining
the presence of multiple species in the sample
  • The dissociation curves show typical primerdimer
    formation. The specific product is shown with a
    Tm of 87 C, whereas the primerdimer has a
    characteristically lower Tm of 79 C.
  • Primerdimers will be most prevalent in NTC (no
    template control) wells and sample wells
    containing low concentrations of template.

56
  • Advantages of Real time PCR
  • Sensitive assay, highly quantitative, and highly
    reproducible
  • Can detect as few as 5 molecules
  • Good Excellent dynamic range, linear over several
    orders of magnitude
  • Useful for diagnostic purposes
  • Disadvantages of Real time PCR
  • Expensive (instruments are gt30-100K,materials
    are also expensive)
  • Medium throughput (10s to 100s of genes)
  • Can pick up RNA carryover or contaminating RNA
    leading to false positives
  • Need a skillful person
  • www.protocol-online.net/molbio/RNA/

57
Useful link for Realtime qPCR
58
Basic research within the biomedical sciences
  • Real-time PCR has impacted a wide variety of
    topics of study, and the examples of gene
    expression analyses are innumerable.
  • Real-time PCR can be used for
  • Genotyping knockout, knockin, and transgenic
    mouse models
  • Determining the efficacy of gene knockdown and
    delivery methods in animals or cell culture
    systems.

59
Basic research within the biomedical sciences
  • Real-time PCRs capability
  • Allelic discrimination
  • Detection of SNPs that may predispose
    individuals to particular diseases can be
    determined in populations.

60
Molecular diagnostic
  • Viral load and identification of virus
  • Bacterial infection early detection specific
    targeting and better treatment
  • detect Mycobacterium tuberculosis, Legionella
    pneumophila, Listeria monocytogenes, and
    Neisseria gonorrhoea.
  • Antibiotic resistance Staphylococcus aureus,
    Staphylococcus epidermidis, Helicobacter pylori,
  • Enterococcus faecalis, and Enterococcus faecium

61
Smallpox virus, cowpox virus, vaccinia virus,
and monkeypox virus can infect humans.
Classified in the single genus Orthopoxvirus.Maj
or problem they are closely related and it is
very difficult to differentiate between these
viruses?Need of level 4 lab to manipulate the
specimens!problem associated with transport

Application of Real time PCR in medicine
Smallpox virus
62
Differentiation of Orthopoxvirus by real time
PCR melting curve
63
  • Mycobacterium haemophilum and Lymphadenitis in
    Children
  • Lesla E.S. Bruijnesteijn van Coppenraet, Edward
    J. Kuijper, Jerome A. Lindeboom, Jan M.
    Prins, and Eric C.J. ClaasLeiden University
    Medical Center, Leiden, the Netherlands and
    Academic Medical Centre, Amsterdam, the
    Netherlands
  • Infections associated with Mycobacterium
    haemophilum
  • are underdiagnosed because specific culture
    methods
  • required for its recovery are not applied
    routinely. Using
  • polymerase chain reaction (PCR) technology on
    fine needle
  • aspirates and biopsied specimens from 89 children
    with
  • cervicofacial lymphadenitis, we assessed the
    importance of
  • M. haemophilum. Application of a Mycobacterium
  • genusspecific real-time PCR in combination with
    amplicon
  • sequencing and a M. haemophilumspecific PCR
    resulted
  • in the recognition of M. haemophilum as the
    causative
  • agent in 16 (18) children with cervicofacial
    lymphadenitis.
  • M. avium was the most frequently found species
    (56),
  • and M. haemophilum was the second most commonly
    recognized
  • pathogen. Real-time PCR results were superior to
  • culture because only 9 (56) of the 16 diagnosed

64
Mayo Clinical Microbiology Laboratory
  • Decreased the analytic turnaround time for six
    different pathogens from a range of 114 days by
    traditional methods to 3050 min using real-time
    PCR and has obtained these results with similar
    or better sensitivities .

65
Clinical oncology
  • To quantify chromosomal translocations
  • To quantify their fusion gene transcripts
    present in a patient sample for use in the
    determination of minimum residual disease (MRD)
    or disease progression
  • detect MRD in patients by measuring the
    AML-1/MTG8 fusion gene product of acute
    myeloblastic leukemia ALL gene rearrangement,
    interferon response.

66
Summary Steps involved in planning RT-QPCR
67
Real Time PCR
  • Powerful tool in diagnostic
  • Bacterial infection
  • Viral infection
  • Cancer
  • Chronic diseases
  • Discovery research, in particular medical
  • Understanding molecular events behind
    physiological, pathological manifestations
  • Drug discovery

68
Definition terminology
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