REVIEW ARTICLE

Methods in molecular cardiology: quantitative real-time polymerase chain reaction J.P.G. Sluijter, M.B. Smeets, G. Pasterkamp, D.P.V. de Kleijn

Changes in mRNA expression levels occur during physiological and pathological processes in the cardiovascular system. An increase in DNA transcription results in increased mRNA levels and will subsequently cause an increase in levels of proteins that regulate processes inside and outside the cell. To determine alterations in mRNA levels, traditional methods such as Northern blotting and ribonuclease protection assay can be used; however, large amounts of RNA are necessary and the methods are very labour intensive. In this review, we focus on one of the newest advancements in polymerase chain reaction (PCR) technology, realtime or quantitative PCR, using small amounts of RNA to determine expression levels. We discuss the technique in general and describe two different approaches. (NethHeartJ2003;11:401-4.) Key words: polymerase chain reaction, real-time, quantitative

uring physiological and pathological processes

expression levels of mRNA and proteins are changed to achieve biological effects. Studying mRNA levels will help us to understand the underlying processes, since alteration of mRNA levels often precedes a change in protein levels which is necessary to obtain the desired effect. Traditional methods to determine mRNA expression levels are Northern blotting, ribonuclease protection and semiquantitative or competitive polymerase chain reaction (PCR). PCR J.P.G. Sluliter. M.B. Smeets.

0. Paterkamp. D.P.V. de Klqln. Experimental Cardiology Laboratory, Heart Lung Centre Utrecht, University Medical Centre, Utrecht. Interuniversity Cardiology Institute of the Netherlands (ICIN), Utrecht. Address for correspondence: D.P.V. de Kleijn. E-mail: [email protected]

Nctherlands Heart Journal, Volume 11, Number 10, October 2003

has been used in the past as a technique in molecular biological research to explore differential expression of genes" 2 and is based on the principle that PCR reactions will amplify target DNA sequences to such a level that expression levels and nudeotide sequences, for example, can be determined. Here we focus on one ofthe newest advancements in PCR technology: real-time or quantitative-PCR3 4

The basic principle of real-time PCR is the classical PCR reaction, as reviewed by Sonnemans et al.2 In short, the polymerase chain reaction (PCR), developed by Dr Mullis,56 is a rapid in vitro enzymatic amplification of a specific deoxyribonucleic acid (DNA) region (figure 1). The basic principle of PCR is formation of complementary strands. First, the doublestranded template is denatured to form single-stranded molecules (denaturation). Next, synthetic oligonucleotide primers, which flank the target DNA sequence that has to be amplified, hybridise to the single-stranded template and serve as the starting point of the DNA polymerase (annealing). Finally, DNA polymerase synthesises a complementary singlestranded DNA matching the template in a process that is called extension. Repeated denaturation - annealing - extension cycles accumulate the target sequence exponentially. The reaction continues as long as there is an excess of primers, oligonucleotides and an active polymerase and occurs in only one direction. The classical PCR reaction needs analysis afterwards, which is time-consuming, not quantitative and contaminations can occur. Modifications of the classical PCR reaction are needed to use this technique for quantifying mRNA levels. An essential step in determining mRNA levels is the conversion of RNA to a complementary DNA template (cDNA) by the enzyme reverse transcriptase. This cDNA is now used as the template in the PCR reaction. Real-time PCR makes it possible to quantify the amount oftarget mRNA at the begiiining of the PCR reaction. This technique is very accurate, reproducible and sensitive, with a high throughput so that con401

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tamination is reduced to a minimum. In real-time PCR reactions, fluorescent molecules are used to monitor the reaction while amplification is taking place. For this, non-specific reporter molecules (e.g. SYBR-green, ethidium bromide) as well as specific probes (e.g. molecular beacons, TaqMan4, dual-oligo FRET pairs, Scorpions4/Amplifluor4) can be used. 1. The use of non-specific molecules will be fast and cheap and can be used for every primer set, but controls are needed to demonstrate specificity ofthe monitored fluorescent signal. 2. A more sensitive and specific way to monitor the amplified signal is the use of specific hybridising probes; however setting up this assay is more time-consuming and more expensive.

Non-specific fluorescent quantification Using non-specific fluorescent molecules is a relatively simple technique that can be applied for any PCR reaction. It is based on the binding of intercalating dyes that can insert between two adjacent base pairs in a molecule of double-stranded DNA, distorting the architecture of the double helix. Only the dye bound to DNA will give a fluorescent emission signal, and not the free dyes that are in the mixture (figure 2). During the PCR cycles more and more doublestranded DNA is formed, thus the amount of the dye that can bind will increase, resulting in an increased fluorescent signal. The increase in fluorescent signal is caused by all amplified DNA products formed in the reactions, which are not necessarily specific for the desired product. When using this approach, primers must be selected carefully and PCR reactions must be optimised to avoid formation of non-desired products. Post-PCRcorrection is sometimes necessary to obtain accurate quantitative information. With this kind of DNA amplification the expression ofseveral genes can be measured relatively quickly and easily, without the use of specific probes. 402

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Figure 2. Non-specific intercalating dye binding during amplification. Dyesbind to double-stranded DNA and when bound they will give afluorescent emission signal. Thefree dye willgive no signal when excited. During amplification more and more double-stranded product is formed and an increasing amount of dye can bind, ths increasing the obsredfluorescent signal.

Smeets et al.7 used the non-specific molecule SYBRgreen in real-time PCR to quantify haptoglobin mRNA expression levels after balloon dilation in rabbits. Haptoglobin is an acute-phase glycoprotein, mainly produced in the liver and secreted in the serum. But increasing evidence shows that haptoglobin is also produced in extrahepatic tissues, for example the arterial wall. Haptoglobin is thought to play a role in cell migration and arterial restructuring. In this study they found an early increase in haptoglobin mRNA levels, which could be measured in the pico-gram range. Furthermore they discovered that arterial haptoglobin consists of an unique set of glycoforms, with other oligosaccharides attached, compared with haptoglobin produced in the liver. Alterations in glycosylation are known to modify function, for example, so it is conceivable that arterial haptoglobin has a different function than liver-produced haptoglobin.

Speclflc fluorescent quanticatIon To ensure that the increase in fluorescent signal is more sensitive and specific, fluorescent oligonucleotide hybridisation probes can be used. Setting up this kind of analysis will cost more time, but for very large studies it will be worth the investment. One strategy often used is the TaqMan assay (figure 3A). In this assay, the probe is labelled at the 5' end with a fluorescent reporter and at the 3' end with a quencher molecule and is complementary to the target DNA. The quencher molecule absorbs the energy emitted by a fluorophore and emits the energy at a different wavelength, reducing the fluorescence of the fluorophore. When the probe is still intact the reporter and quencher are near each other and the fluorescence of the 5' reporter is absorbed by the 3' quencher and no signal is detected. PCR amplification ofthe target DNA will increase the number of probes that hybridise with the complementary template. Netherlnds Heart Journal, Volume 11, Number 10, October 2003

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During the extension phase of a reaction, the Taqpolymerase cleaves the labelled probe which is hybridised. The reporter molecule is then released from the quencher and the fluorescent signal can be detected. Accumulation of the released reporter molecules during the amplification cycles results in an increasing fluorescent signal and is correlated to the amount of the target DNA present. Another hybridisation probe is the molecular beacon, which is a hairpin-loop shaped single-stranded oligonucleotide (figure 3B). It consists of a probe sequence, homologous to the target sequence, but is flanked by complimentary arm sequences, which are homologous to one another but not to the target sequence. One arm contains a fluorescent reporter and the other a non-fluorescent quencher. Similar to the Taqman assay, the unbound reporter and quencher are next to each other and the fluorescent signal will be captured by the quencher. When the probe binds to the target sequence this will result in a separation of the reporter and quencher molecule and the fluorescent signal can be monitored. Because more and more probes will bind to the DNA, the fluorescent signal will increase during amplification. Ortiz-Pallard6 et al.8 used real-time PCRto identify the PiZ (proteinase inhibitor) mutations and to determine heterozygous and homozygous carrier status in whole blood and from paraffin-embedded specimens. A mutation in the cx1-antitrypsin (AAT), with the structural gene locus encoding AAT called Pi, resulting in AAT deficiency is a common inherited cause of emphysema and cirrhotic liver disease. Current diagnostic techniques are time-consuming and have a limited accuracy. Ortiz-Pallard6 et al. compared the results of the RT-PCR with single-strand conformational polymorphism (SSCP) and direct DNA 4 Amplifiation plot and standard curvegenerated with the iCylkr iQ software (Bio-rad laboratories, Inc). A. 7le amplification plot displays the observed increased fluoresentsignalfor eacb weU (one line rpresents one well). 7Te thr6sold isdeterminedby the backgreund signalfound in the first cycles, and tbe cycle in wbich tbe observed fluoreszence rises above this thrshold is called the treshold ccle andwill be used in the analysis. B. The standard curve isgeneratedfrom the targtreferncesamples and the DNA concentration of the unknowns are calculated. The cormlation ceficint is a measure ofthe accuracy ofthe data compared with the generated curve. The slope of the curve represents tbe PCR efficiency.

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Netherlands Heart Journal, Volume 11, Number 10, October 2003

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Methods in molecular cardiology: quantitative real-time polymerase chain reaction

sequencing, which are currently used. They found that the results ofthe RT-PCRwere confirmed by the other two techniques in all cases, but allowed a higher throughput level. Setting up a quantitative PCR Amplification in a real-time PCRwiII increase the target DNA and therefore also the observed fluorescent signal. The higher the starting amount of the target DNA in the sample the sooner a significant increase in the fluorescent signal will be registered. The cycle number observed when the fluorescent signal rises above background levels is used to quantify the amount of DNA, and is called the threshold cycle. There is a linear relationship between the threshold cycle during real-time PCR and the log of the starting amount of a template. Quantification of the target will be performed by comparison with a standard curve ofthe target reference standard. Computerised analysis of the data is needed to quantify the starting amounts in the samples. A grid of the reaction plate is installed to let the computer know which part of the signal represents the well that is recorded by the CCD camera. The amplification plot (figure 4) displays the relative fluorescence for each well at every cycle. To determine threshold cycles and the standard curve, a PCR baseline subtraction is needed. This will correct for the fluorescent background level, the noise in the samples, and is automatically calculated from the optimal baseline cydes for each well individually or can be applied manually. After this, the standard curve can be generated and the amount of DNA in the unknown samples can be calculated. From the created standard curve, a correlation coefficient and the efficiency can be calculated. The correlation coefficient tells you something about the accuracy of the experimental data compared with the expected values, thus how well a sample fits to the created standard curve. The efficiency of the reactions is directly related to the slope of the standard curve and provides information about the PCRreaction itself. An efficiency of 100% represents a doubling of the template after each cycle and corresponds with the theoretically perfect PCR reaction. Equal efficiencies in the different PCR reactions are needed to be able to compare different PCR runs.

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Concluding remarks Using real-time PCR, it is possible to monitor different expression levels of different mRNAs in one reaction (real-time multiplex gene expression) when using different probes with different fluorescent signals. Hybridisation probes that do not cleave can identify mutations, even a single-base mutation, within the PCRproduct. Hybridisation probes can also determine the distribution of genotypes within a population compared with known genotypes. The real-time PCR can also be used to quickly verify the data obtained by microarray. Quantitative, or real-time, PCR is an accurate, sensitive and high throughput technique that can be used for several purposes. The goal of the study determines which kind of tools are used to obtain the fluorescent data. Some experience is needed to analyse these data and interpret the results, but it will be worth the investment. U

Acknowledgement We thank Eddy van Collenburg (Bio-Rad Laboratories B.V.) for his help with the figures. References 1 2

Smeets MB, Pasterkamp G, Kleijn DPV de. Methods in molecular cardiology: suppression subtraction hybridisation. Neth HeartJ 2003;11:260-4. Sonnemans DGP, Wmdt LJ de, Muinck ED de, Doevendans PA. Methods in molecular cardiology: the polymerase chain reaction.

Neth HeartJ2002;10:412-8. Higuchi R, Fockler C, Dollinger G, Watson R Kinetic PCR analysis: real-time monitoring of DNA amplification reactions. Biotechnology (N Y) 1993;11:1026-30. 4 Heid CA, Stevens J, Livak KJ, Williams PM. Real time quantitative PCGR Genome Res 1996;6:986-94. 5 Mullis KB, Faloona F. A specific synthesis of DNA in vitro via a polymerase catalysed chain reaction. Methods Enzymol 1987;155: 335-50. 6 Mullis KB, Faloona FA, ScharfSJ, Saiki RK, Horn GT, Erlich HA. Specific enzymatic amplification of DNA in vitro: the polymerase chain reaction. Cold SpingHarborSympQuantBiol 1986;51:26373. 7 Smeets MB, Sluijter JPG, Donners MPC, Velema E, Heeneman S, Pasterkamp G, et al. Increased arterial expression of a glycosylated haptoglobin isoform after balloon dilation. Cardiovasc Res 2003;58:689-95. 8 Ortiz-Pallardo ME, Zhou H, Fischer HP, Neuhaus T, Sachinidis A, Vetter H, et al. Rapid analysis of alphalantitrypsin PiZ genotype by a real-time PCR approach. JMol Med 2000;78:212-6. 3

Netherlands Heart Journal, Volume 11, Number 10, October 2003

Methods in molecular cardiology: quantitative real-time polymerase chain reaction.

Changes in mRNA expression levels occur during physiological and pathological processes in the cardiovascular system. An increase in DNA transcription...
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