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.
1Quantification 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
-
3Why 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.
4Why 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
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6Real 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.
7Real 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.
8A 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.
9How the signal of Real time PCR is quantified
- The signal measured during these PCR cycles is
used to plot the threshold. - The threshold is calculated as 10 times the
standard deviation of the average signal of the
baseline fluorescent signal. - 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. - The Ct is defined as the fractional PCR cycle
number at which the fluorescent signal is greater
than the minimal detection level.
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12Three 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
13Three main chemistries are used in Real time PCR
- 1- Intercalating dyes
- SYBR-Green
14Three main chemistries are used in Real time PCR
- 1- Intercalating dyes
- SYBR-Green
15Three 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.
16LUX (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.
17LUX (light Upon eXtension) Primers
Invitrogen Web site
18Three 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.
19TaqMan 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.
20TaqMan probe
21Platforms 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.
22Heating 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.
23Air 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.
24Designing 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
25Conti..
- 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
26Conti...
- -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
27Isolation 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
28RNA 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
29RNA 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. -
30RNA 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
31Primers and amplicon for inhibitors assessment
and 3-5assay
32RNA quality assay
33Effect of Primers concentration on QPCR
Nolan T et al, 2006
34cDNA priming
- Oligo dT
- suppose that the RNA is intact.
- non suitable if looking for
- Splice variants
- Sequences long 3 UTR
- Non poly genes
35cDNA 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.
36cDNA 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
38Steps involved in planning RT-QPCR
39RT-QPCR experimental workflow
40RT-qPCR
41Cont...
42Cont...
43Conti
44In 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.
-
45Ct 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
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48How the signal of Real time PCR is quantified
- The signal measured during these PCR cycles is
used to plot the threshold. - The threshold is calculated as 10 times the
standard deviation of the average signal of the
baseline fluorescent signal. - 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. - The Ct is defined as the fractional PCR cycle
number at which the fluorescent signal is greater
than the minimal detection level.
49Comparative 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.
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51Acceptable Standard curve
52Unacceptable standard curve
53Dissociation 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.
54Dissociation curves are useful for determining
the presence of multiple species in the sample
55Dissociation 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/
57Useful link for Realtime qPCR
58Basic 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.
59Basic 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.
60Molecular 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
61Smallpox 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
62Differentiation 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
64Mayo 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 .
65Clinical 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.
66Summary Steps involved in planning RT-QPCR
67Real 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
68Definition terminology