Real-Time PCR Applications Critical Factors for

Integrated Solutions — Real-Time PCR Applications

Critical Factors for Successful Real-Time PCR New

40

Slope = –3.59

CT

35 30 25 20 15 –4

–2

0

2

Log ng total RNA

Rn

3.0

2.0

1.0 0.5 0 0

10

20 30 Cycle number

40

50

W W W. Q I A G E N . C O M

Contents

Introduction

4

Detection of PCR products in real time

5

SYBR Green I

5

Fluorescently labeled sequence-specific probes

5

QuantiProbes

6

TaqMan probes

7

FRET probes

7

Molecular Beacons

7

Dyes used for fluorogenic probes in real-time PCR

8

Methods in real-time PCR

9

Two-step and one-step RT-PCR

9

DNA as template in real-time PCR

9

Basic terms used in real-time PCR

11

Baseline and threshold settings

11

Quantification of target amounts

2

14

Quantification

14

Absolute quantification

14

Relative quantification

15

Determining amplification efficiencies

17

Different amplification efficiencies

17

Guidelines for relative quantification with different amplification efficiencies

18

Comparable amplification efficiencies

19

Guidelines for relative quantification with comparable amplification efficiencies

19

Comparative method or ∆∆CT method of relative quantification

20

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Critical Factors for Successful Real-Time PCR 01/2004

Contents

Guidelines for relative quantification using ∆∆CT method

21

Endogenous reference genes

21

Guidelines for successful real-time RT-PCR: reverse transcription

23

1. Choice of RT primers

23

2. Universal priming method for real-time, two-step RT-PCR

24

3. Effect of RT volume added to two-step RT-PCR

24

4. Choice of reverse transcriptase

25

5. One-step RT-PCR

27

Guidelines for successful real-time PCR: amplification

28

1. Primer design

28

2. Effect of cations and hot start on real-time PCR specificity

29

3. Probe-based detection in real-time PCR

32

4. Genomic DNA in real-time RT-PCR

36

QuantiTect Gene Expression Assays

38

QuantiTect Custom Assays

39

Easy-to-use QuantiProbe Design Software

39

Appendix

42

RNA template preparation and stabilization

42

Isolation of RNA from whole blood

42

RNeasy Protect system

42

Determining concentration and purity of nucleic acids

42

Storage of RNA

43

Ordering Information


45

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3

Introduction Real-time PCR and RT-PCR are highly sensitive techniques enabling amplification and quantification of a specific nucleic acid sequence with detection of the PCR product in real time. Quantification of DNA, cDNA, or RNA targets can be easily achieved by determination of the cycle when the PCR product can first be detected. This is in contrast with endpoint detection in conventional PCR, which does not enable accurate quantification of nucleic acids. Real-time PCR is highly suited for a wide range of applications, such as gene expression analysis, determination of viral load, detection of genetically modified organisms (GMOs), SNP genotyping, and allelic discrimination. In this guide, we provide information on the basic principles of real-time PCR, terms used in real-time PCR, and factors influencing the performance of real-time PCR assays. In addition, we describe the advantages and disadvantages of different reaction chemistries as well as providing answers to frequently asked questions and guidelines for successful results. Examples of the spectrum of research currently being carried out are also included. We extend our thanks to those who have contributed to this project and hope that it may provide a useful guide to successful real-time PCR for researchers everywhere.

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Detection of PCR products in real-time

SYBR Green Principle

Real-time PCR and RT-PCR allow accurate quantification of starting

SYBR Green

amounts of DNA, cDNA, and RNA targets. Fluorescence is measured during each cycle, which greatly increases the dynamic range of the

Primer

reaction, since the amount of fluorescence is proportional to the amount of PCR product. PCR products can be detected using either fluorescent

3'

5'

dyes that bind to double-stranded DNA or fluorescently labeled sequence-specific probes. SYBR Green I

Primer 3'

5'

SYBR® Green I binds all double-stranded DNA molecules, emitting a fluorescent signal of a defined wavelength on binding (Figure 1). The

excitation

excitation and emission maxima of SYBR Green I are at 494 nm and Primer

521 nm, respectively, and are compatible for use with any real-time cycler. Detection takes place in the extension step of real-time PCR. Signal intensity increases with increasing cycle number due to the accumulation of PCR product. Use of fluorescent dyes enables analysis of

emission

3'

5'

Figure 1 Principle of SYBR Green I based detection of PCR products in real-time PCR.

many different targets without having to synthesize target-specific labeled probes. However, nonspecific PCR products and primer-dimers will also contribute to the fluorescent signal. Therefore, high PCR specificity is required when using SYBR Green I. Fluorescently labeled sequence-specific probes Fluorescently labeled probes provide a highly sensitive and specific method of detection, as only the desired PCR product is detected. However, PCR specificity is also important when using sequence-specific probes. Amplification artifacts, such as nonspecific PCR products and primer-dimers may also be produced, which can result in reduced yields of the desired PCR product. Competition between the specific product and reaction artifacts can compromise assay sensitivity and efficiency. The following section discusses different probe chemistries.


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QuantiProbe™ Principle

QuantiProbes

Fluorophore Quencher

MGB Superbases

QuantiProbes, included in QuantiTect® Gene Expression Assays and QuantiTect Custom Assays (see pages 38–40), are sequence-specific fluorescently labeled probes with a fluorophore at the 3' end, and a

A

nonfluorescent quencher and minor groove binder at the 5' end (Figure 2).

excitation Primer

MGB™ technology leads to increased probe stability and allows the use of shorter probes. QuantiProbes form a random structure in solution,

3'

which facilitates efficient quenching of the fluorescent signal. When the

5'

QuantiProbe hybridizes to its target sequence during the PCR annealing B

step, the fluorophore and quencher separate and a fluorescent signal is

emission excitation

Primer

of PCR product present in the reaction at a given time point, enabling

5'

3'

generated. The fluorescent signal is directly proportional to the amount sensitive and accurate quantification of target sequences. The MGB prevents hydrolysis of the QuantiProbe by the 5' → 3' exonuclease

C excitation

activity of Taq DNA polymerase, and the probe is simply displaced from the template during the extension step of PCR.

Primer 3'

5'

Modified bases known as Superbases, included in the QuantiProbe and

Figure 2 Principle of QuantiProbes in quantitative, real-time PCR. A When not bound to its target sequence, the QuantiProbe forms a random structure in solution. The proximity of the fluorescent reporter with the quencher prevents the reporter from fluorescing. B During the PCR annealing step, the QuantiProbe hybridizes to its target sequence. This separates the fluorescent dye and quencher resulting in a fluorescent signal. The amount of signal is proportional to the amount of target sequence, and is measured in real time to allow quantification of the amount of target sequence. C During the extension step of PCR, the probe is displaced from the target sequence, bringing the fluorophore and quencher into closer proximity, resulting in quenching of fluorescence.

primers, together with the MGB at the 5' end of the QuantiProbe, allow successful detection of target sequences that may not have worked well in previous real-time PCR assays, such as AT-rich or GC-rich sequences. Superbases are analogs of the corresponding naturally occurring bases. SuperA™ and SuperT™ form strong bonds with their unmodified complementary bases in the target sequence. The stability of the base pair is comparable to that of a GC base pair. SuperA in the QuantiProbe forms three hydrogen bonds with its complementary T base in the target sequence. In contrast, SuperT in the QuantiProbe forms only two hydrogen bonds with the unmodified A base in the target sequence, but stability of the base pair is increased by improved base stacking.

TaqMan Probe Principle Fluorophore

SuperG™ inhibits the formation of tetrad aggregates from a run of several Quencher

Cleaved nucleotides

Gs in a row and enables probe design for GC-rich sequences. The modified bases, together with the MGB, enable shorter probes to be used due to the increased Tm. This allows use of primers and probe at

A excitation

predefined sequences, such as splice sites, while still delivering optimal real-time PCR performance — something that may not be possible with

Primer 3'

5'

B excitation

emission

Primer 3'

6

5'

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other dual-labeled probes or FRET probes.

Figure 3 Principle of TaqMan probes in quantitative, real-time PCR. A Both the TaqMan probe and the PCR primers anneal to the target sequence during the PCR annealing step. The proximity of the fluorescent reporter to the quencher prevents the reporter from fluorescing. B During the PCR extension step, Taq DNA polymerase extends the primer. When the enzyme reaches the probe, its 5' → 3' exonuclease activity cleaves the fluorescent reporter from the probe. The fluorescent signal from the free reporter is measured.


TaqMan probes

FRET Probe Principle

TaqMan® probes, are sequence-specific oligonucleotide probes carrying

F1 Fluorophore (donor)

F2 Fluorophore (acceptor)

a fluorophore and a quencher dye (Figure 3). The fluorophore is attached at the 5' end of the probe and the quencher dye is located at the 3' end.

excitation

A

During the combined annealing/extension phase of PCR, the probe is Primer

cleaved by the 5' → 3' exonuclease activity of Taq DNA polymerase,

F1 F2

separating the fluorophore and the quencher dyes. This results in detectable fluorescence that is proportional to the amount of accumulated

3'

5'

PCR product.

excitation

B

FRET probes PCR with fluorescence resonance energy transfer (FRET) probes, such as

transfer

Primer

F1

emission

F2

3'

5'

LightCycler® hybridization probes, uses two labeled oligonucleotide probes that bind to the PCR product in a head-to-tail fashion (Figure 4).

excitation

C

When the two probes bind, their fluorophores come into close proximity,

F1

allowing energy transfer from a donor to an acceptor fluorophore. Therefore, fluorescence is detected during the annealing phase of PCR and is proportional to the amount of PCR product. FRET probes usually carry dyes that are only compatible for use on the LightCycler machine. As the FRET system uses two primers and two probes, good design of the primers and probes is critical for successful results. Molecular Beacons Molecular Beacons are dual-labeled probes with a fluorophore attached at the 5' end and a quencher dye attached at the 3' end. The probes are designed so that the ends are complementary. When the probe is in solution, the two ends of the probe hybridize and form a stem-loop structure with the fluorophore and quencher in close proximity (Figure 5).

F2

Primer 3'

5'

Figure 4 Principle of FRET probes in quantitative, real-time PCR. A When not bound to their target sequence, no fluorescent signal from the acceptor fluorophore is detected. B During the PCR annealing step, both FRET probes hybridize to the target sequence. This brings the donor and acceptor fluorophore into close proximity, allowing energy transfer between the fluorophores and resulting in a fluorescent signal from the acceptor fluorophore that is detected. The amount of signal is proportional to the amount of target sequence, and is measured in real time to allow quantification of the amount of target sequence. C During the extension step of PCR, the probes are displaced from the target sequence and the acceptor fluorophore is no longer able to generate a fluorescent signal.

This efficiently quenches the fluorescent signal. When the probe binds to

Molecular Beacon Principle

the target sequence, the stem opens and the fluorophore and quencher Fluorophore

separate. This generates a fluorescent signal in the annealing step that is proportional to the amount of PCR product. With Molecular Beacons,

Quencher

A

successful probe design can be difficult.

Primer excitation 3'

5'

B Figure 5 Schematic diagram of the principle of Molecular Beacons in quantitative, real-time PCR. A When not bound to its target sequence, the Molecular Beacon forms a hairpin structure. The proximity of the fluorescent reporter with the quencher prevents the reporter from fluorescing. B During the PCR annealing step, the Molecular Beacon probe hybridizes to its target sequence. This separates the fluorescent dye and reporter, resulting in a fluorescent signal. The amount of signal is proportional to the amount of target sequence, and is measured in real time to allow quantification of the amount of target sequence. C During the extension step of PCR, the probe is displaced from the target sequence, bringing the fluorophore and quencher into closer proximity, resulting in quenching of fluorescence.


Primer

excitation

emission

3'

5' excitation

C Primer 3'

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5'

7

Table 1. Dyes Commonly Used for Quantitative, Real-Time PCR

Dye

Excitation maximum (nm)

Dyes used for fluorogenic probes in real-time PCR Except for SYBR Green I detection, which uses a double-stranded DNA

Emission maximum (nm)*

binding molecule, all methods for quantitative, real-time PCR are based on fluorescently labeled probes. The wide variety of fluorescent dyes

Fluorescein

490

513

Oregon Green

492

517

FAM

494

518

SYBR Green I

494

521

cycler used. The emission spectra of the chosen dyes must also be

TET

521

538

sufficiently distinct from one another. For information on excitation and

JOE

520

548

emission spectra for commonly used fluorescent dyes, see Table 1.

VIC

538

552

Yakima Yellow™

526

552

HEX

535

553

Cy®3

552

570

TAMRA

560

582

Cy3.5

588

604

ROX

587

607

Use a control sample in which the gene of interest is definitely

Texas Red

596

615

expressed. PCR products that span the region to be amplified in

LightCycler-Red 640 (LC640)

625

640

the real-time experiment can also be used as a positive control.

Cy5

643

667

Check by agarose gel electrophoresis that the amplification

Cy5.5

683

707

reaction was successful. The quality of the starting template and

available makes real-time multiplex PCR possible, providing the dyes are compatible with the excitation and detection criteria of the real-time

?

My sample does not give a fluorescent signal. How can I decide whether this is because the PCR did not work or because the target is not expressed?

the integrity of the reagents can be determined by amplifying a * Emission spectra may vary depending on the buffer conditions.

housekeeping gene, such as GAPDH or HPRT.

?

What are Superbases and the MGB for? Superbases are analogs of the naturally occurring bases. SuperA and SuperT stabilize probe and primer binding within AT-rich regions. SuperG inhibits the formation of tetrad aggregates from a run of several Gs in a row in the primers and probe. The MGB stabilizes the template–probe hybrid and, compared with conventional probes, allows the use of shorter probe sequences. The combination of Superbases with the MGB enables design of real-time RT-PCR assays at pre-defined sequences — even for difficult templates.

?

Do I need to calibrate my real-time cycler if I want to use Yakima Yellow? The emission maximum of Yakima Yellow (552 nm) is almost identical to that of the fluorescent dye VIC. Therefore, the channel and filter settings for VIC can also be used for Yakima Yellow.

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Methods in real-time PCR Two-step and one-step RT-PCR For analysis of gene expression levels or viral load, the RNA first needs to be transcribed into cDNA using a reverse transcriptase. Reverse transcriptases are enzymes generally derived from RNA-containing retroviruses. RT-PCR can take place in a two-step or one-step reaction. With two-step RT-PCR, the RNA is first reverse transcribed into cDNA using oligo-dT primers, random oligomers, or gene-specific primers. An aliquot of the reverse-transcription reaction is then subsequently added to the real-time PCR. In two-step RT-PCR, it is possible to choose between different types of RT primers, depending on experimental needs. Use of oligo-dT primers or random oligomers for reverse transcription means that several different transcripts can be analyzed by PCR from one RT reaction. In addition, precious RNA samples can be immediately transcribed into more stable cDNA for later use and long-term storage. In one-step RT-PCR — also referred to as one-tube RT-PCR — both reverse transcription and amplification take place in the same tube, with reverse transcription preceding PCR. This is possible due to specialized reaction chemistries and cycling protocols (see pages 23–27). The fast procedure enables rapid processing of multiple samples and is easy to automate. The reduced number of handling steps results in high reproducibility from sample to sample and minimizes the risk of contamination since less manipulation is required. The advantages of each method are given below. Two-step RT-PCR

One-step RT-PCR

■ Multiple PCRs from one RT reaction

■ Easy handling

■ Flexibility with RT primer choice

■ Fast procedure

■ Enables long-term storage of cDNA

■ High reproducibility ■ Low contamination risk

DNA as template in real-time PCR

Reliable SNP Genotyping with Small Amounts of Template

In contrast with RNA, which requires conversion into cDNA, purified genomic DNA or plasmid DNA can be directly used as starting template

Allele 1 homozygote

Allele 2 homozygote

in real-time PCR. Not only can genomic DNA be quantified in real-time

Heterozygote

No DNA

7.00

also be used for qualitative analysis, such as single nucleotide

5.50

polymorphism (SNP) detection. SNP analysis involves the detection of single nucleotide changes using two probes labeled with different fluorophores. One probe is specific for the wild-type allele, the other for the mutant allele (Figure 6). Real-time PCR is highly suited for the detection of small sequence differences, such as SNPs and viral variants.

Allele 2

PCR, for example, in detection of bacterial DNA or GMOs, but it can

4.50 3.00 1.50 0.13 0.26

1.00

1.80

2.60

3.40

Allele 1 Figure 6 Real-time PCR was carried out using the QuantiTect Probe PCR Kit and dual-labeled (TaqMan) probes for the CPY2D6*4 SNP, using 2 ng or 5 ng DNA template. The allelic discrimination plots clearly indicate the homozygotes for each allele and the heterozygotes.


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Application data Quenched FRET assay enables detection of single base mutations

Wild type

Quenched FRET assays are similar to FRET assays except that the

H63D heterozygote H63D/S65C heterozygote

decrease in energy of the donor fluorophore is measured instead of the 5

designed to detect the single base mutations H63D and S65C. The

4

QuantiTect Probe PCR Kit enabled highly sensitive detection of wild type and mutant sequences (Figure 7).

Fluorescence -dF/dT

increase in energy of the acceptor fluorophore. FRET probes were

3 2 Threshold

1 0 –1 50

55

60 65 Temperature (°C)

70

75

Figure 7 Following real-time PCR using the QuantiTect Probe PCR Kit on the Rotor-Gene™ system, amplification products were subjected to melting curve analysis. (Data kindly provided by T. Kaiser, Corbett Research, Australia.)

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Basic terms used in real-time PCR

Typical Amplification Plot

A number of basic terms are used in real-time PCR analyses and are

Sample A Sample B

Data are displayed as sigmoidal-shaped amplification plots (when using a linear scale), in which the fluorescence is plotted against the number of cycles (Figure 8).

Fluorescence

briefly described in the following section.

Log-linear phase Threshold

Baseline and threshold settings

when analyzing several genes in parallel or when using probes carrying different reporter dyes, the baseline and threshold setting must be

10

0

the raw data must be analyzed and baseline and threshold values set. When different probes are used in a single experiment, for example,

Baseline

0

Before gene expression levels can be quantified in real-time PCR studies,

20

30

40

Cycle number Figure 8 Amplification plots showing increases in fluorescence from 2 samples (A and B). Sample A contains a higher amount of starting template than sample B.

adjusted for each probe. Furthermore, analysis of different PCR products from a single experiment using SYBR Green I detection requires baseline

Correct Baseline and Threshold Settings are Important for Accurate Quantification

and threshold adjustments for each individual assay. Guidelines are given below for data analysis. For more information on data analysis,

A 24

real-time cycler.

20

Baseline: The baseline is the noise level in early cycles, typically measured

16

between cycles 3 and 15, where there is no detectable increase in

∆Rn

refer to the recommendations provided by the manufacturer of the

12 8

fluorescence due to amplification products. The number of cycles used to

4

calculate the baseline can be changed and should be reduced if high

-1

template amounts are used or if the expression level of the target gene is high (Figure 9A). To set the baseline, view the fluorescence data in the linear scale amplification plot. Set the baseline so that growth of the amplification plot begins at a cycle number greater than the highest

16 ∆Rn

20

8

Background: This refers to nonspecific fluorescence in the reaction, for

4

example, due to inefficient quenching of the fluorophore or the presence

-1

removed by the software algorithm of the real-time cycler. Reporter signal: Fluorescent signal that is generated during real-time PCR by either SYBR Green I or by a fluorescently labeled sequence-

31

37

1

7

18 25 13 Cycle number

31

37

12

obtained from amplification products.

Green I. The background component of the signal is mathematically

18 25 13 Cycle number

24

individually for each target sequence. The average fluorescence value

of large amounts of double-stranded DNA template when using SYBR

7

B

baseline cycle number (Figure 9B). The baseline needs to be set detected within the early cycles is subtracted from the fluorescence value

1

Figure 9 A Amplification product becomes detectable within the baseline setting of cycles 6 to 15 and generates a wavy curve with the highest template amount. B Setting the baseline within cycles 6 to 13 eliminates the wavy curve. The threshold is set at the beginning of the detectable log-linear phase of amplification.

specific probe.


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Normalized reporter signal (Rn): This is the emission intensity of the reporter dye divided by the emission intensity of the passive reference dye measured in each cycle. Passive reference dye: On some real-time PCR instruments, the fluorescent dye ROX serves as an internal reference for normalization of the fluorescent signal. It allows correction of well-to-well variation due to pipetting inaccuracies and fluorescence fluctuations. Its presence does not interfere with real-time PCR assays, since it is not involved in PCR and has an emission spectrum completely different from fluorescent dyes commonly used for probes. Threshold: The threshold is adjusted to a value above the background and significantly below the plateau of an amplification plot. It must be placed within the linear region of the amplification curve, which represents the detectable log-linear range of the PCR. The threshold value should be set within the logarithmic amplification plot view to enable easy identification of the log-linear phase of the PCR. If several targets are used in the real-time experiment, the threshold must be set for each target. Threshold cycle (CT) or crossing point: This is the cycle at which the amplification plot crosses the threshold, i.e., at which there is a significant detectable increase in fluorescence. The CT serves as a tool for calculation of the starting template amount in each sample (see pages 14–22 for more information). ∆CT value: The ∆CT value describes the difference between the CT value of the target gene and the CT value of the corresponding endogenous reference gene, such as a housekeeping gene: ∆CT = CT (target gene) – CT (endogenous reference gene) ∆∆CT value: The ∆∆CT value describes the difference between the average ∆CT value of the sample of interest (e.g., stimulated cells) and the average ∆CT value of a reference sample (e.g., unstimulated cells). The reference sample is also known as the calibrator sample and all other samples will be normalized to this when performing relative quantification: ∆∆CT = average ∆CT (sample of interest) – average ∆CT (reference sample) For more information on ∆CT and ∆∆CT see “Quantification of target amounts”, pages 14–22. Endogenous reference gene: This is a gene whose expression level should not differ between samples, such as a housekeeping or maintenance gene. Comparison of the CT value of a target gene with that of the endogenous reference gene allows the gene expression level of the target gene to be normalized to the amount of input RNA or cDNA (see ∆CT value). This is done without determining the exact amount of template used in the reaction. The use of an endogenous reference gene corrects for variation in RNA content, variation in reverse-transcription efficiency, possible RNA degradation or presence of inhibitors in the RNA sample, variation in nucleic acid recovery, and differences in sample handling. Genes commonly used as references are shown in Table 2.

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Table 2. Housekeeping Genes Commonly Used as Endogenous References Gene name

Relative expression level*

18S ribosomal RNA

++++

Glyceraldehyde-3-phosphate dehydrogenase

+++

β-Actin, cytoplasmic

+++

β-2-microglobulin

+++

Phosphoglycerate kinase 2

+++

Peptidylprolyl isomerase A (cyclophilin A)

+++

Ribosomal protein, large, PO

+++

Hypoxanthine phosphoribosyl transferase 1

++

β-Glucuronidase

+

TATA box binding protein

+

Transferrin receptor

+

* ”+” indicates relative abundance of the transcripts.

Calibrator sample: This is a reference sample used in relative quantification. For example, RNA isolated from a cell line or tissue, to which all other samples are compared to determine the relative expression level of a gene. The calibrator sample usually has a stable expression ratio of target gene to endogenous reference gene. No template control (NTC): A control reaction that contains all essential components of the amplification reaction except the template. This enables detection of contamination. RT minus control: RNA preparations may contain residual genomic DNA, which may be detected in real-time PCR if assays are not designed to only detect and amplify RNA (see pages 36–37). DNA contamination can be detected by performing a control reaction in which no reverse transcription takes place. Standard: Sample of known concentration or copy number used to construct a standard curve. Standard curve: To generate a standard curve, CT values/crossing points of different standard dilutions are plotted against the logarithm of input amount of standard material. The standard curve is commonly generated using a dilution series of at least 5 different concentrations of the standard. Each standard curve should be checked for validity, with the value for the slope falling between –3.3 to –3.8. Standards are ideally measured in triplicate for each concentration. Standards which give a slope differing greatly from these values should be discarded. Efficiency and slope: The slope of a standard curve provides an indication of the efficiency of the real-time PCR. A slope of –3.322 means that the PCR has an efficiency of 1, or 100%, and the amount of PCR product doubles during each cycle. A slope of less than –3.322 (e.g., –3.8) is indicative of a reaction efficiency