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REAL-TIME PCR
M. Tevfik Dorak, MD,
PhD
Dorak MT (Ed): Real-Time PCR (Advanced Methods Series). Oxford:
Taylor & Francis, 2006
Glossary of Terms Used in Real-Time PCR
PowerPoint
Presentation on Real-Time PCR
Webinars on qPCR
(by Mikael Kubista)
Real-time
reverse-transcriptase (RT) PCR quantitates the initial amount of the template
most specifically, sensitively and reproducibly, and is a preferable
alternative to other forms of quantitative RT-PCR that detect the amount of
final amplified product at the end-point 1 2 (Freeman,
1999; Raeymaekers,
2000). Real-time PCR monitors the fluorescence emitted during the
reaction as an indicator of amplicon production during each PCR cycle (ie, in
real time) as opposed to the endpoint detection 3,4 (Higuchi,
1992; Higuchi,
1993). The real-time progress of the reaction can be viewed in some
systems. Real-time PCR does not detect the size of the amplicon and thus does
not allow the differentiation between DNA and cDNA amplification, however, it
is not influenced by non-specific amplification unless SYBR Green is used (see
below). Real-time PCR quantitation (qPCR) eliminates post-PCR processing of PCR
products (which is necessary in competitive RT-PCR). This helps to increase
throughput and reduce the chances of carryover contamination. In comparison to
conventional RT-PCR, real-time PCR also offers a much wider dynamic range of up
to 107-fold (compared to 1000-fold in conventional RT-PCR). Dynamic
range of any assay determines how much target concentration can vary and still
be quantified. A wide dynamic range means that a wide range of ratios of target
and normalizer can be assayed with equal sensitivity and specificity. It
follows that the broader the dynamic range, the more accurate the quantitation.
The real-time PCR system is based
on the detection and quantitation of a fluorescent reporter 5,6 (Lee,
1993; Livak,
1995). This signal increases in direct proportion to the amount of PCR
product in a reaction. By recording the amount of fluorescence emission at each
cycle, it is possible to monitor the PCR reaction during exponential phase
where the first significant increase in the amount of PCR product correlates to
the initial amount of target template. The higher the starting copy number of
the nucleic acid target, the sooner a significant increase in fluorescence is
observed. A significant increase in fluorescence above the baseline value
measured during the 3-15 cycles indicates the detection of accumulated PCR
product.
A fixed fluorescence threshold is
set significantly above the baseline that can be altered by the operator. The
parameter CT (threshold cycle) is defined as the cycle number at
which the fluorescence emission exceeds the fixed threshold. There are three main fluorescence-monitoring systems for DNA amplification 7 (Wittwer,
1997a): (1) hydrolysis probes; (2) hybridizing probes (see Hybridization
Probe Chemistry); and (3) DNA-binding agents 8,9 (Wittwer,
1997b; van
der Velden, 2003). Hydrolysis probes include TaqMan
probes 10 (Heid, 1996), molecular
beacons 11-15 (Mhlanga,
2001; Vet,
2002; Abravaya,
2003; Tan, 2004; Vet
& Marras, 2005) and scorpions (further
details) 16-18 (Saha,
2001; Solinas,
2001; Terry,
2002). They use the fluorogenic 5' exonuclease activity of Taq
polymerase to measure the amount of target sequences in cDNA samples (see also 19 Svanvik,
2000 for light-up probes).
TaqMan probes are oligonucleotides
longer than the primers (20-30 bases long with a Tm value of 10 oC higher)
that contain a fluorescent dye usually on the 5' base, and a quenching dye
(usually TAMRA) typically on the 3' base (TaqMan MGB probes have a
non-fluorescent quencher and minor groove binder at the 3’ end). When
irradiated, the excited fluorescent dye transfers energy to the nearby
quenching dye molecule rather than fluorescing (this is called FRET = Förster
or fluorescence resonance energy transfer) 20,21 (Hiyoshi,
1994; Chen,
1997). Thus, the close proximity of the reporter and quencher prevents
emission of any fluorescence while the probe is intact. TaqMan probes are
designed to anneal to an internal region of a PCR product. When the polymerase
replicates a template on which a TaqMan probe is bound, its 5' exonuclease
activity cleaves the 5’ end of probe which contains the reporter dye 22 (Holland,
1991). This ends the activity of quencher (no FRET) and the reporter
dye starts to emit fluorescence which increases in each cycle proportional to
the rate of probe cleavage. Accumulation of PCR products is detected by
monitoring the increase in fluorescence of the reporter dye (note that primers
are not labeled). TaqMan assay uses universal thermal cycling parameters and
PCR reaction conditions. Because the cleavage occurs only if the probe
hybridizes to the target, the origin of the detected fluorescence is specific
amplification. The process of hybridization and cleavage does not interfere
with the exponential accumulation of the product. One specific requirement for
fluorogenic probes is that there be no G at the 5' end. A 'G' adjacent to the
reporter dye quenches reporter fluorescence even after cleavage. Well-designed
TaqMan probes require very little optimization (see a list of SNP500 Cancer Validated
TaqMan Allelic Discrimination Assays).
Molecular beacons also contain
fluorescent (FAM, TAMRA, TET, ROX) and quenching dyes (typically DABCYL) at
either end but they are designed to adopt a hairpin structure while free in
solution to bring the fluorescent dye and the quencher in close proximity for
FRET to occur. They have two arms with complementary sequences that form a very
stable hybrid or stem. The close proximity of the reporter and the quencher in
this hairpin configuration suppresses reporter fluorescence. When the beacon
hybridizes to the target during the annealing step, the reporter dye is
separated from the quencher and the reporter fluoresces (FRET does not occur).
Molecular beacons remain intact during PCR and must rebind to target every
cycle for fluorescence emission. This will correlate to the amount of PCR
product available. All real-time PCR chemistries allow detection of multiple
DNA species (multiplexing) by designing each probe/beacon with a spectrally
unique fluor/quench pair, or if SYBR green is used by melting curve analysis.
By multiplexing, the target(s) and endogenous control can be amplified in
single tube for qPCR purposes. For examples, see 23-31 (Bernard, 1998;
Vet, 1999; Lee,
1999; Donohoe,
2000; Read,
2001; Grace,
2003; Vrettou,
2004; Rickert, 2004;
Persson, 2005.
With Scorpion primer/probes,
sequence-specific priming and PCR product detection is achieved using a single
oligonucleotide. The Scorpion probe maintains a stem-loop configuration in the
unhybridized state. The fluorophore is attached to the 5' end and is quenched
by a moiety coupled to the 3' end. The 3' portion of the stem also contains
sequence that is complementary to the extension product of the primer. This
sequence is linked to the 5' end of a specific primer via a non-amplifiable
monomer. After extension of the Scorpion primer, the specific probe sequence is
able to bind to its complement within the extended amplicon thus opening up the
hairpin loop. This prevents the fluorescence from being quenched and a signal
is observed (see also How It Works)
The cheaper alternative is the
double-stranded DNA binding dye chemistry, which quantitates the amplicon
production (including non-specific amplification and primer-dimer complex) by
the use of a non-sequence specific fluorescent intercalating agent (SYBR-green
I or ethidium bromide). It does not bind to ssDNA. SYBR green is a fluorogenic
minor groove binding dye that exhibits little fluorescence when in solution but
emits a strong fluorescent signal upon binding to double-stranded DNA 32 (Morrison,
1998). Disadvantages of SYBR green-based real-time PCR include the
requirement for extensive optimization. Furthermore, non-specific
amplifications require follow-up assays (melting point or dissociation curve
analysis) for amplicon identification 33 (Ririe,
1997). The method has been used in HFE-C282Y genotyping 26 (Donohoe,
2000). Another controllable problem is that longer amplicons create a
stronger signal (if combined with other factors, this may cause CDC camera
saturation, see below). Normally SYBR green is used in singleplex reactions,
however when coupled with melting curve analysis, it can be used for multiplex
reactions 34 (Siraj,
2002).
The threshold cycle or the CT
value is the cycle at which a significant increase in DRn is
first detected (for definition of DRn, see
below and glossary).
The threshold cycle is when the system begins to detect the increase in the
fluorescent signal associated with an exponential growth of PCR product during
the log-linear phase. This phase provides the most useful information about the
reaction (certainly more important than the end-point). The slope of the
log-linear phase reflects the amplification efficiency (Eff). Eff can be calculated by the formula:
Eff = 10(-1/slope)
–
1
The
efficiency of the PCR should be 90 - 100% (– 3.6 > slope > – 3.1)
(Stratagene
Slope to Efficiency Calculator). A number of variables can affect the
efficiency of the PCR 35-37 (Bustin, 2004; Wong, 2005;
Yuan,
2006). These factors include length of the amplicon, secondary
structure and primer quality. Although valid data can be obtained that fall
outside of the efficiency range, the qRT-PCR should be further optimized or
alternative amplicons designed (see Efficiency
Determination Page by Pfaffl). For the slope to be an
indicator of real amplification (rather than signal drift), there has to be an
inflection point. This is the point on the growth curve when the log-linear phase
begins. It also represents the greatest rate of change along the growth curve.
(Signal drift is characterized by gradual increase or decrease in fluorescence
without amplification of the product.) The important parameter for quantitation
is the CT. The higher the initial amount of genomic DNA, the sooner
accumulated product is detected in the PCR process, and the lower the CT
value. The threshold should be placed above any baseline activity and within
the exponential increase phase (which looks linear in the log transformation).
Some software allows determination of the cycle threshold (CT) by a
mathematical analysis of the growth curve. This provides better run-to-run
reproducibility. A CT value of 40 or higher means no amplification
and this value cannot be included in the calculations. Besides being used for
quantitation, the CT value can be used for qualitative analysis as a
pass/fail measure.
Relative gene expression comparisons work best when the gene expression of the chosen endogenous/internal control is more abundant and remains constant, in proportion to total RNA, among the samples. By using an invariant endogenous control as an active reference, quantitation of an mRNA target can be normalized for differences in the amount of total RNA added to each reaction. For this purpose, the most common choices are 18S RNA, GAPDH (glyceraldehyde-3-phosphate dehydrogenase) and b-actin. Because the 18S mRNA does not have a poly-A tail, cDNA synthesis using oligo-dT should not be used if 18S RNA will be used as a normalizer. The issue of the choice of a normalizer has been reviewed by Suzuki et al. 38 (Suzuki, 2000). The authors recommend caution in the use of GAPDH as a normalizer as it has been shown that its expression may be upregulated in proliferating cells. They recommend b-actin as a better active reference. GAPDH is severely criticized as a normalizer by others too 39-41 (Bustin SA, 2000; Dheda, 2004; Aerts, 2004). GAPDH is particularly an unpopular choice in cancers because of its increased expression in aggressive cancers 42 (Goidin, 2001). Caution should also be exercised when 18S RNA is used as a normalizer as it is a ribosomal RNA species (not mRNA) and may not always represent the overall cellular mRNA population. Since the chosen mRNA species should be proportional to the amount of input RNA, it may be best to use a combination as normalizer. It is desirable to validate the chosen normalizer for the target cell or tissue. It should be expressed at a constant level at different time points by the same individual and also by different individuals at the target cell or tissue (for example, peripheral blood lymphocytes) 40 (Dheda, 2004). This aim can be achieved by the ABI's TaqMan Human Endogenous Control Plate or TATAA Biocenter's Endogenous Control Gene Panel which evaluate the expression of select housekeeping genes. Our own experience