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REAL-TIME PCR
M. Tevfik Dorak, MD, PhD
exonuclease activity of Taq polymerase to measure the amount of target sequences in cDNA
samples (see also 19 Svanvik, 2000 for light-up probes).
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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
or a non-fluorescent quencher (NFQ)) typically on the 3' base (TaqMan MGB probes have a NFQ 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 = Frster 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. To increase specificity of a
TaqMan probe and have shorter probes, MGB or locked nucleic acid (LNA) probes can be used with
equal efficiency (Kutyavin, 2000; Letertre, 2003; Johnson, 2004; Ugozzoli, 2004). See Glossary
for LNA, MGB, NFQ; see also a list of SNP500 Cancer Validated TaqMan Allelic Discrimination
Assays).
Molecular beacons also contain fluorescent (FAM, TAMRA, TET, ROX) and quenching dyes (typically
DABCYL or BHQ) 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. Consideration of excitation, emission and absorption spectral relationships between
acceptor and donor flurophores (LightCycler probes), reporter and quencher flurophores (TaqMan
probes) and multiplexing is important and discussed in BHQ Brochure (Biosearch Technologies)
and Marras SA, 2006.
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
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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) (see Agilent Efficiency
Calculator from the Slope). 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 loglinear 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 rRNA, GAPDH (glyceraldehyde-3-phosphate
dehydrogenase) and b-actin, but not necessarily the most suitable choices (Radonic, 2004).
Because the 18S RNA 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 18S RNA is an overabundant RNA species, and if used
as a normalizer, the CT values are likely to be around 10-12. Any sample that has 18S CT values >15
may be considered poor quality. 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 bactin as a better active reference (but see Dheda, 2004). 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 cancer studies because of its increased expression in aggressive cancers 42
(Goidin, 2001). Caution should also be exercised when 18S rRNA is used as a normalizer as it is a
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ribosomal RNA species (not mRNA) and may not always represent the overall cellular mRNA
population. Since it is abundantly expressed, 18S rRNA yields very small (<15) CT values, which is
not desirable, especially when the target gene has a low expression level. This problem can be
overcome by using Ambions QuantumRNA Technology. 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 TaqMan Human Endogenous Control Plate, geNorm kit or TATAA
Biocenter Endogenous Control Gene Panel which evaluate the expression of select
housekeeping genes. Our own experience 43 (Sabek, 2002) showed that b-actin or 18S RNA are
reasonable choices as normalizers for the peripheral blood mononuclear cells, whereas GAPDH
performed worst in transplant monitoring studies. Similar concerns on the choice of normalizers in
transplant monitoring have also been expressed by others 44 (Gibbs, 2003). Surveys of tumor cell
lines or tissues reported the worst results with GAPDH while beta-glucuronidase (GUS) and 18S
rRNA 41 (Aerts, 2004) or HPRT 45 (de Kok, 2004) were the best choices for this target. It is
important to choose a normalizer whose expression will remain constant under the experimental
conditions designed for the target gene 46 (Schmittgen, 2000; Dheda, 2005). Another study found
that for robust conclusions the use of multiple internal controls is the best choice (18S rRNA and
cyclophilin) for kidney mRNA expression studies 47 (Schmid, 2003). The strategy of using multiple
normalizer genes depending on the cell and tissue type is validated for general use 48
(Vandesompele, 2002). (See Ambion: 18S RNA as an Internal Control; Ambion: GAPDH, b-actin,
cyclophilin, 18S RNA as internal controls; EXPOLDB: The most constantly expressed
housekeeping genes; algorithms to select the best endogenous controls: geNORM
(Vandesompele, 2002), NormFinder (Andersen, 2004), and qBasePlus (Hellemans, 2007)). See
also GeneInvestigator for normalizer selection (Hruz, 2011).
Multiplex TaqMan assays can be performed using multiple dyes with distinct emission wavelengths.
Available dyes for this purpose are FAM, TET, VIC and JOE (the most expensive). TAMRA is reserved
as the quencher on the traditional TaqMan probes and ROX as the passive reference. For best
results, the combination of FAM (target) and VIC (endogenous control) is recommended (they have
the largest difference in emission maximum) whereas JOE and VIC should not be combined. It is
important that if the dye layer has not been chosen correctly, the machine will still read the other dye's
spectrum. For example, both VIC and FAM emit fluorescence in a similar range to each other and
when doing a single dye, the wells should be labeled correctly. In the case of multiplexing, the spectral
compensation for the post run analysis should be turned on (on ABI 7700:
Instrument/Diagnostics/Advanced Options/Miscellaneous). Activating spectral compensation
improves dye spectral resolution.
One-step real-time RT-PCR performs reverse transcription and PCR in a single buffer system and in
one tube. In two-step RT-PCR, these two steps are performed separately in different tubes.
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specific priming. The primer conditions are the same for SYBR Green assays,
6. Maximum amplicon size should not exceed 400 bp (ideally 50-150 bases). Smaller amplicons give
more consistent results because PCR is more efficient and more tolerant of reaction conditions (the
short length requirement has nothing to do with the efficiency of 5' nuclease activity),
7. The probes should not have runs of identical nucleotides (especially four or more consecutive Gs),
G+C content should be 30-80%, there should be more Cs than Gs, and not a G at the 5' end. The
higher number of Cs produces a higher DRn (this feature may require manual check). The choice of
probe should be made first,
8. To avoid false-positive results due to amplification of contaminating genomic DNA in the cDNA
preparation, it is preferable to have primers spanning exon-exon junctions in the cDNA sequence.
This way, genomic DNA will not be amplified,
9. If a TaqMan probe is designed for allelic discrimination, the mismatching nucleotide (the
polymorphic site) should be in the middle of the probe rather than at the ends,
10. Use primers that contain dA nucleotides near the 3' ends so that any primer-dimer generated is
efficiently degraded by AmpErase UNG (mentioned in p.9 of the manual for EZ RT-PCR kit; P/N
402877). If primers cannot be selected with dA nucleotides near the ends, the use of primers with 3'
terminal dU-nucleotides should be considered.
See also ABgene Dual Labeled Probe Design Guide.
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cycles before the CT value for the most abundant sample). Threshold default is 10 standard
deviations of the background signal above mean fluorescence generated during baseline cycles in ABI
instruments. This threshold value will be used to calculate the CT values for each sample in the run.
For a useful discussion of this matter, see the ABI Tutorial on Setting Baselines and Thresholds
and Real-Time PCR: Understanding CT. (Interestingly, this issue is best discussed in the manual
for TaqMan Human Endogenous Control Plate.)
If the results do not make sense, check the raw spectra for a possible CDC camera saturation during
the run. Saturation of CDC camera may be prevented by using optical caps rather than optical
adhesive cover. It is also more likely to happen when SYBR Green I is used, when multiplexing and
when a high concentration of probe is used.
For manuals and other educational material about Idaho Technology instruments, see Support
page. See also a qPCR Protocol for SmartCycler at Qiagen website, and another Protocol for
SmartCycler II; Critical Factors for Successful Real-time PCR by Qiagen.
Interpretation of results
At the end of each reaction, the recorded fluorescence intensity is used for the following calculations
by the software of the system used:
Rn+ is the Rn value of a reaction containing all components (the sample of interest); Rn- is the Rn
value detected in NTC (baseline value). DRn is the difference between Rn+ and Rn-. It is an indicator
of the magnitude of the signal generated by the PCR (DRn may be called RFU=relative fluorescence
unit in some instruments). It is the DRn plotted against cycle numbers that produces the amplification
curves and gives the CT value.
There are different approaches to quantitate the amount of template 50 (Livak, 2001):
1. Absolute standard curve method: Absolute quantification determines the input copy number of
the transcript of interest, usually by relating the PCR signal to a standard curve. In this method, a
standard curve is first constructed from an RNA of known concentration. This curve is then used as a
reference standard for extrapolating quantitative information for mRNA targets of unknown
concentrations. cDNA plasmids are the preferred standards for absolute quantitation. This method
has been used to estimate cytokine concentrations 51 (Giulietti, 2001), CMV 52-55 (Kearns, 2001a;
Kearns, 2001b; Kearns, 2002; Mengelle, 2003), HIV 56 (Gibellini, 2004) and other viral loads 57
(Niesters, 2001), 16 (Saha, 2001). See Bustin, 2000 for a review 39; and Absolute Quantification
Page by Pfaffl.
2. Relative standard method (relative fold change): In this method, one of the experimental
samples is the calibrator, or 1x sample. Each of the normalized target values is divided by the
calibrator normalized target value to generate the relative expression levels. Target quantity is
determined from the standard curve and divided by the target quantity of the calibrator. The calibrator
is the 1x sample, and all other quantities are expressed as an n -fold difference relative to the
calibrator. The calibrator is usually the expression level at baseline and the experimental samples are
those collected after treatment or some intervention. The calibrator should be available at large
enough quantities to be included in each run. See Relative Quantification Page by Pfaffl.
3. Comparative threshold (CT) method (DDCT): This method uses no known amount of standard
but compares the relative amount of the target sequence to any of the reference values chosen and
the result is given as relative to the reference value (such as the expression level of resting
lymphocytes or a standard cell line or in comparison to the baseline value). For the CT calculation to
be valid, the efficiency of the target amplification and the efficiency of the reference amplification must
be approximately equal. A sensitive method for assessing if two amplicons have the same efficiency
is to look at how CT varies with template dilution. Before using the DDCT method for quantitation, a
validation experiment is performed to demonstrate that efficiencies of target and reference are
approximately equal. Serial dilutions of the target and normalizer are prepared and real-time PCR is
run in separate tubes. The CT values for each dilution of the target and the normalizer are obtained
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and their difference for each dilution is calculated (DCT). Then, a plot of log input (like from 0.01 ng to
100 ng) amount versus DCT is prepared (minimum three different dilutions). If the efficiencies of the
two amplicons are approximately equal, the plot of log input amount versus DCT has a slope of
approximately zero (the absolute value of the slope of log input amount vs CT should be < 0.1). This
method has been used in monitoring the immune system activity after transplantation 43 (Sabek,
2002). See Livak & Schmittgen, 2001 for a review 50; and ABI-7700 User Bulletin #2 for the details
of quantitation methods.
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increase, and for decreased expression it will be something like 2-3 = 1/8 of the reference level.
Microsoft Excel can be used to do these calculations by simply entering the CT values (there is an
online ABI tutorial on the use of spread sheet programs to produce amplification plots; the TaqMan
Human Endogenous Control Plate protocol also contains detailed instructions on using MS Excel
for real-time PCR data analysis). Statistical assessment of the difference of DDCT values from 0 can
be achieved by a number of methods, the simplest being the t-test and Wilcoxon test (Yuan, 2006). A
more accurate method of relative quantification using the relative expression ratio is presented by
Pfaffl 58 (Pfaffl, 2001).
The quantification methods are outlined in the ABI User Bulletins. The Bulletins #2 and #5 are most
useful for the general understanding of real-time PCR and quantification.
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* Monitoring chimerism after hematopoietic stem cell transplantation 106-109 (Elmaagacli, 2002;
Alizadeh, 2002; Thiede, 2004; Harries, 2004)
* Monitoring minimal residual disease after hematopoietic stem cell transplantation 9,106,110-112
(Elmaagacli, 2002; Cilloni, 2002; Sarris, 2002; Gabert, 2003; Van der Velden, 2003)
* Determination of gene dosage and zygosity 113-115 (Bubner, 2004; Barrois, 2004; Chen, 2006;
Szilagyi, 2006; Wu, 2007; Parajes, 2007 & 2008)
* Genotyping by fluorescence melting-curve analysis (FMCA) or high-resolution melting (HRM)
analysis 26,116-123 (von Ahsen 2000; Donohoe, 2000; Lyon, 2001; Waterfall & Cobb, 2002;
Bennett, 2003; Wittwer, 2003; Zhou, 2005; Palais, 2005; Chou, 2005) or specific probes/beacons
11,17,124-127 (Tapp, 2000; Mhlanga, 2001; Solinas, 2001; Song, 2002; Gupta, 2004; reviewed in
Lareu, 2004). LNA or MGB probes can be used allelic discrimination too (Kutyavin, 2000; Letertre,
2003; Johnson, 2004; Ugozzoli, 2004; Gibson NJ, 2006; Shen, 2009; Schleinitz, 2011)
- Trisomies 128 (Zimmermann, 2002) and single-gene copy numbers 129-132 (Bieche, 1998;
Mocellin, 2003, Barrois, 2004; Linzmeier, 2005)
- Microdeletion genotypes 133-136 (Laurendeau, 1999; Kariyazono, 2001; Covault, 2003; Coupry,
2004; Rose-Zerilli, 2009 (GST deletion))
- Haplotyping 137,138 (Von Ahsen, 2004; Pont-Kingdon & Lyon, 2005)
- Quantitative microsatellite analysis 139 (Ginzinger, 2000)
- DNA pooling and quantitative allelic discrimination 140-142 (Barcellos, 2001; Abbas, 2004;
Quesada, 2004; Gibson NJ, 2006)
- Prenatal diagnosis / sex determination using single cell isolated from maternal blood 143-145
(Hahn, 2000; Bischoff, 2002; Bischoff, 2003) or fetal DNA in maternal circulation 144,146 (Bischoff,
2002; Hwa, 2004)
- Prenatal diagnosis of hemoglobinopathies 29,147,148 (Kanavakis, 1997; Vrettou, 2003; Vrettou,
2004)
- Intraoperative cancer diagnostics 149 (Raja, 2002)
* Linear-after-the-exponential (LATE)-PCR: a new method for real-time quantitative analysis of target
numbers in small samples, which is adaptable to high throughput applications in clinical diagnostics,
biodefense, forensics, and DNA sequencing 150 (Sanchez, 2004).
Full References Cited
Internet links
Idaho Technology LightScanner & FilmArray
ROCHE LightCycler Online Bio-Rad: CFX96 Stratagene-Agilent qPCR System
Corbett/Qiagen Rotor-Gene
Cepheid: Smart Cycler / GeneXpert Eppendorf Mastercycler
Applied Biosystems Sequence Detection Systems
Bio-Rad Real-Time PCR Applications Guide
Critical Factors for Successful Real-time PCR (Qiagen)
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ABI User Bulletins ABI-PRISM 7700 Application Notes 7900HT 7000 Compendium
SNP500Cancer Validated Allelic Discrimination Assay List (including TaqMan Protocols)
Available Real-Time PCR Platforms BioCompare Horizon Press
1st International qPCR Symposium & Application Workshop (qPCR 2009)
qPCR Guide (EuroGentec)
Scorpion Technology Molecular Beacons Light-Up Probes (1) (2)
D-LUX Designer LNA Probes LNA Primers (Exiqon OligoDesign) Exiqon ProbeLibrary
DesignMyProbe at Sigma-Aldrich
Fluidigm (high-throughput qPCR, CNV, genotyping)
TaqMan Gene Expression Assays
Products at TATAA BioCenter & GenEx
Primer-Probe and Beacon Design Program & Demo by Premier
RT-PCR PrimerBank RT-PCR Primer DataBase RT-PCR Primer Sets
AlleleID Pathogen Detection Primer & Probe Design Tool by Premier Biosoft International
BioSearch RealTimeDesign Software (QuickStart Guide)
Quantitative PCR Primer Database - QPPD (NCI)
PrimerDesign InVitroGene: Custom Primers-OligoPerfect Designer
Frequently Asked Questions (Real-Time PCR Primers)
Applied Biosystems: Real-Time PCR Animated Tutorials (for beginners)
Transcript of a Webcast on Real-Time PCR Applications (Bio.Com)
Biocompare Tutorials:
Tools and Technologies for Real-Time PCR & Fast PCR (text)
Sigma-Aldrich qPCR Webinars
Invitrogene Molecular Probes Handbook
Roche LightCycler Literature and Technical Notes
QiaGen Handbooks on SYBR Green Detection Systems and RT-PCR
Ambion TechNotes on Real-Time PCR
Full qPCR Protocol (Nolan, Hands & Bustin, Nature Protocols, 2006) (PDF)
Gene Quantification Page by Michael W Pfaffl & Directory Page
Real-Time PCR Tutorial (South Carolina University)
Real-Time PCR: Short Course (University of Texas)
Real-Time PCR Handbook (University of Illinois at Chicago)
Troubleshooting & Optimization Guide (Thermo Scientific)
Real-Time PCR Seminar (NIEHS) & Review by Nigel Walker
Real-Time PCR in Infectious Diseases (PPT) & (PDF) by Theo Sloots
Real Time PCR & Quantitation Lecture by Ian MacKay
Essentials of Real-Time PCR Lecture by Man Bock Gu
Q-PCR Training @ TATAA BioCenter
PCR and Real Time PCR Links Real-Time PCR Literature
Links at PCRlinks.com Links at Protocol Online
Review of real-time PCR in mRNA Quantitation (Wong & Medrano, 2005)
Five Questions on qPCR & How It Works (The Scientist)
Statistics and Gene Expression Analysis BioInformatics in Real-Time PCR
qPCR Data Analysis Presentation (RB Lanz, 2008)
Q-GENE for data processing
geNORM (Vandesompele, 2002) NormFinder (Andersen, 2004) qBasePlus (Hellemans, 2007)
BestKeeper for determination of stable housekeeping genes (Download)
REST for Relative Expression Software Tool (REST-2008 / Corbett)
CAmpER - Real-time PCR Analysis Software
Peirson, 2003 (DART-PCR) (Download)
SNPman (User Guide) for TaqMan allelic discrimination assay genotype calling
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for the ABI7300, LC480 and Biorad CFX platforms (Konopac, 2011)
Real Time PCR Special Issues:
METHODS: Dec 2001, Vol.25, Issue 4 & April 2010, Vol.50, Issue 4
CLINICA CHIMICA ACTA: Jan 2006, Vol.363, Issue 1-2
HUMAN MUTATION: June 2009, Vol.30, Issue 6 (High-Resolution Melting Technology)
Books:
Real-Time PCR (Dorak MT)
A-Z of Quantitative PCR (Bustin S)
Rapid Cycle Real-Time PCR-Methods and Applications (Wittwer Hahn, Kaul)
Real-Time PCR: An Essential Guide (Edwards, Logan, Saunders)
Real-Time PCR: Current Technology and Applications (Logan, Edwards, Saunders)
Real-time PCR in Microbiology (MacKay IM)
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