qPCR_Handbook

i REAL-TIME PCR: FROM THEORY TO PRACTICE ii This handbook is printed on recycled paper. Put the odds in your favor with SuperScript® RT. Why chance your research with anything other than the most reliable, consistent reverse transcriptase in the industry? Choose SuperScript® III RT for robust first-strand synthesis and great cDNA yields. Engineered to be RNase H– and thermostable up to 60°C, SuperScript® I I I RT delivers best-in-class cDNA synthesis in every single reaction, combining maximum yield with maximum sensitivity. You get full-length cDNA without degrading rare RNA transcripts. And whether you’re cloning, doing micro- array analysis, or monitoring gene expression with qRT-PCR, SuperScript® III RT always delivers the results you need to move forward—the first time, every time. You can bet on it. Go for complete reliability at www.invitrogen.com/superscript. 2008 © Invitrogen Corporation. All rights reserved. These products may be covered by one or more Limited Use Label Licenses (see the Invitrogen catalog or our website, www.invitrogen.com). Engineered to be RNase H– and incredibly thermostable, SuperScript® III RT delivers robust first-strand synthesis with both great cDNA yield and ultimate sensitivity, whatever the size of your mRNA. iii www.invitrogen.com Contents Basic principles of real-time PCR . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 Overview of real-time PCR . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4 Overview of qPCR and qRT-PCR components . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 Real-time PCR analysis terminology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8 Real-time PCR fl uorescence detection systems . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14 Melting curve analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16 Use of passive reference dyes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18 Contamination prevention. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19 Multiplex real-time PCR . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20 Internal controls and reference genes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20 Real-time PCR instrument calibration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21 iv Important Licensing Information: These products may be covered by one or more Limited Use Label Licenses (see Invitrogen catalog or our website, www.invitrogen.com). By use of these products you accept the terms and conditions of all applicable Limited Use Label Licenses. All products are for research use only. CAUTION: Not intended for human or animal diagnostic or therapeutic uses. Assay design . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .22 Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22 Real-time PCR assay types . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22 Amplicon and primer design considerations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23 Nucleic acid purifi cation and quantitation. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27 Reverse transcription considerations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30 Controls . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 34 Normalization methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 34 Using a standard curve to assess effi ciency, sensitivity, and reproducibility . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 38 Data analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .40 Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40 High-resolution melt curve (HRM) analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 47 Multiplex real-time PCR analysis. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 51 Troubleshooting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .55 Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 55 Frequently asked questions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 67 3 www.invitrogen.com 3 Section IBasic principles of real-time PCR Basic principles of real-tim e PCR Basic principles of real-time PCR Introduction The polymerase chain reaction (PCR) is one of the most powerful technolo- gies in molecular biology. Using PCR, specifi c sequences within a DNA or cDNA template can be copied, or “amplifi ed”, many thousand- to a million- fold. In traditional (endpoint) PCR, detection and quantitation of the amplifi ed sequence are performed at the end of the reaction after the last PCR cycle, and involve post-PCR analysis such as gel electrophoresis and image analysis. In real-time quantitative PCR (qPCR), the amount of PCR product is measured at each cycle. This ability to monitor the reaction during its exponential phase enables users to determine the initial amount of target with great precision. PCR theoretically amplifi es DNA exponentially, doubling the number of molecules present with each amplifi cation cycle. The number of cycles and the amount of PCR end-product can theoretically be used to calculate the ini- tial quantity of genetic material (by comparison with a known standard), but numerous factors complicate this calculation. The ethidium bromide staining typically used to quantify endpoint PCR products prevents further amplifi ca- tion, and is only semiquantitative. PCR may not be exponential for the fi rst several cycles, and the reaction eventually plateaus, so the amount of DNA should be measured while the reaction is still in the exponential amplifi cation phase, which can be diffi cult to determine in endpoint PCR. To address these factors, the technique of real-time quantitative PCR was developed. In real-time PCR, the amount of DNA is measured after each cycle by the use of fl uorescent markers that are incorporated into the PCR product. The increase in fl uorescent signal is directly proportional to the number of PCR product molecules (amplicons) generated in the exponential phase of the reaction. Fluorescent reporters used include double-stranded DNA (dsDNA)- binding dyes, or dye molecules attached to PCR primers or probes that are incorporated into the product during amplifi cation. The change in fl uorescence over the course of the reaction is measured by an instrument that combines thermal cycling with scanning capability. By plotting fl uorescence against the cycle number, the real-time PCR instrument generates an amplifi cation plot that represents the accumulation of product over the duration of the entire PCR reaction (Figure 1). 4 Important Licensing Information: These products may be covered by one or more Limited Use Label Licenses (see Invitrogen catalog or our website, www.invitrogen.com). By use of these products you accept the terms and conditions of all applicable Limited Use Label Licenses. All products are for research use only. CAUTION: Not intended for human or animal diagnostic or therapeutic uses. 4 Real-time PCR: from theory to practice The advantages of real-time PCR include: The ability to monitor the progress of the PCR reaction as it occurs in → real time The ability to precisely measure the amount of amplicon at each cycle → An increased dynamic range of detection → The combination of amplifi cation and detection in a single tube, which → eliminates post-PCR manipulations Figure 1—Amplifi cation plots are created when the fl uorescent signal from each sample is plotted against cycle number; therefore, amplifi cation plots represent the accumulation of product over the duration of the real-time PCR experiment. The samples being amplifi ed in this example are a dilution series of the template. Cycle number 0 18 24126 30 36 3816 22104 28 3414 2082 26 32 40 R el at iv e flu or es ce nc e 4,000 3,500 3,000 2,500 2,000 1,500 1,000 500 0 Baseline fluorescence Ct Fluorescence threshold 108 107 106 105 104 103 102 101 NTC Target input Over the past several years, real-time PCR has become the leading tool for the detection and quantifi cation of DNA or RNA. Using these techniques, you can achieve precise detection that is accurate within a two-fold range, and a dynamic range of 6 to 8 orders of magnitude. Overview of real-time PCR This section provides an overview of the steps involved in performing real- time PCR. Real-time PCR is a variation of the standard PCR technique used to quantify DNA or RNA in a sample. Using sequence-specifi c primers, the relative number of copies of a particular DNA or RNA sequence can be deter- mined. By measuring the amount of amplifi ed product at each stage during the PCR cycle, quantifi cation is possible. If a particular sequence (DNA or RNA) is abundant in the sample, amplifi cation is observed in earlier cycles; if the sequence is scarce, amplifi cation is observed in later cycles. Quantifi cation of amplifi ed product is obtained using fl uorescent probes or fl uorescent DNA- binding dyes and real-time PCR instruments that measure fl uorescence while performing temperature changes needed for the PCR cycles. 5 www.invitrogen.com 5 Section IBasic principles of real-time PCR Basic principles of real-tim e PCR qPCR steps There are three major steps that make up a qPCR reaction. Reactions are gen- erally run for 40 cycles. Denaturation1. —The temperature should be appropriate to the poly- merase chosen (usually 95°C). The denaturation time can be increased if template GC content is high. Annealing2. —Use appropriate temperatures based on the calculated melting temperature (Tm) of the primers (5°C below the Tm of the primer). Extension3. —At 70–72°C, the activity of the DNA polymerase is optimal, and primer extension occurs at rates of up to 100 bases per second. When an amplicon in qPCR is small, this step is often combined with the annealing step using 60°C as the temperature. Two-step qRT-PCR Two-step qRT-PCR starts with the reverse transcription of either total RNA or poly(A)+ RNA into cDNA using a reverse transcriptase (RT). This fi rst-strand cDNA synthesis reaction can be primed using random hexamers, oligo(dT), or gene-specifi c primers (GSPs). To give an equal representation of all targets in real-time PCR applications and to avoid the 3´ bias of oligo(dT), it is usually recommended that random hexamers or a mixture of oligo(dT) and random hexamers are used. The temperature used for cDNA synthesis depends on the RT enzyme chosen. Following the fi rst-strand synthesis reaction, the cDNA is transferred to a separate tube for the qPCR reaction. In general, only 10% of the fi rst- strand reaction is used for each qPCR. One-step qRT-PCR One-step qRT-PCR combines the fi rst-strand cDNA synthesis reaction and qPCR reaction in the same tube, simplifying reaction setup and reducing the possibility of contamination. Gene-specifi c primers (GSP) are required. This is because using oligo(dT) or random primers will generate nonspe- cifi c products in the one-step procedure and reduce the amount of product of interest. Overview of qPCR and qRT-PCR components This section provides an overview of the major reaction components and parameters involved in real-time PCR experiments. A more detailed discussion of specifi c components like reporter dyes, passive reference dyes, and uracil DNA glycosylase (UDG) is provided in subsequent sections of this handbook. 6 Important Licensing Information: These products may be covered by one or more Limited Use Label Licenses (see Invitrogen catalog or our website, www.invitrogen.com). By use of these products you accept the terms and conditions of all applicable Limited Use Label Licenses. All products are for research use only. CAUTION: Not intended for human or animal diagnostic or therapeutic uses. 6 Real-time PCR: from theory to practice DNA polymerase PCR performance is often related to the DNA polymerase, so enzyme selec- tion is critical to success. One of the main factors aff ecting PCR specifi city is the fact that Taq DNA polymerase has residual activity at low temperatures. Primers can anneal nonspecifi cally to DNA, allowing the polymerase to syn- thesize nonspecifi c product. The problem of nonspecifi c products resulting from mispriming can be minimized by using a “hot-start” enzyme. Using a hot-start enzyme ensures that no active Taq is present during reaction setup and the initial DNA denaturation step. Reverse transcriptase The reverse transcriptase (RT) is as critical to the success of qRT-PCR as the DNA polymerase. It is important to choose an RT that not only provides high yields of full-length cDNA but also has good activity at high temperatures. High-temperature performance is also very important for tackling RNA with secondary structure or when working with gene-specifi c primers (GSPs). In one-step qRT-PCR, an RT that retains its activity at higher temperatures allows you to use a GSP with a high melting temperature (Tm), increasing specifi city and reducing background. dNTPs It is recommended that both the dNTPs and the Taq DNA polymerase be pur- chased from the same vendor, as it is not uncommon to see shifts of one full threshold cycle (Ct) in experiments that employ these items from separate vendors. Magnesium concentration In qPCR, magnesium chloride or magnesium sulfate is typically used at a fi nal concentration of 3 mM. This concentration works well for most targets; how- ever, the optimal magnesium concentration may vary between 3 and 6 mM. Good experimental technique Do not underestimate the importance of good laboratory technique. It is best to use dedicated equipment and solutions for each stage of the reactions, from preparation of the template to post-PCR analysis. The use of aerosol- barrier tips and screwcap tubes can help decrease cross-contamination problems. To obtain good replicates (ideally, triplicates), a master mix that contains all the reaction components should be prepared. The use of a mas- ter mix reduces the number of pipetting steps and, consequently, reduces the chances of cross-well contamination and other pipetting errors. 7 www.invitrogen.com 7 Section IBasic principles of real-time PCR Basic principles of real-tim e PCR Template Anywhere from 10 to 1,000 copies of template nucleic acid should be used for each real-time PCR reaction. This is equivalent to approximately 100 pg to 1 μg of genomic DNA, or cDNA, generated from 1 pg to 100 ng of total RNA. Excess template may increase the amount of contaminants and reduce effi ciency. If the template is RNA, care should be taken to reduce the chance of genomic DNA contamination. One option is to treat the template with DNase I. Ultrapure, intact RNA is essential for full-length, high-quality cDNA synthesis and accurate mRNA quantifi cation. RNA should be devoid of any RNase contamination, and aseptic conditions should be maintained. To iso- late total RNA, we recommend using either a column-based system such as the PureLink™ RNA Mini Kit, or TRIzol® Plus Reagent. Isolation of mRNA is typi- cally not necessary, although incorporating this step may improve the yield of specifi c cDNAs. To ensure there is no genomic DNA contamination of the RNA preparation, RNA should be treated with amplifi cation-grade DNase I prior to qRT-PCR. Real-time PCR primer design Good primer design is one of the most important parameters in real-time PCR. When designing gene-specifi c real-time PCR primers, keep in mind that the amplicon length should be approximately 80–250 bp, since longer products do not amplify as effi ciently. Optimal results may require a titration of primer concentrations between 50 and 500 nM. A fi nal concentration of 200 nM for each primer is eff ective for most reactions. In general, primers should be 18–24 nucleotides in length. This provides for practical annealing temperatures. Primers should be designed according to standard PCR guidelines. They should be specifi c for the target sequence and be free of internal secondary structure. Primers should avoid stretches of polybase sequences (e.g., poly (dG)) or repeating motifs, as these can hybrid- ize inappropriately to the template. Primer pairs should have compatible melting temperatures (within 5°C) and contain approximately 50% GC content. High GC content results in the formation of stable imperfect hybrids, while high AT content depresses the Tm of perfectly matched hybrids. If possible, the 3´ end of the primer should be rich in GC bases (GC clamp) to enhance annealing of the end that will be extended. The sequences should be analyzed to avoid complementarity and prevent hybridization between primers (primer-dimers). For qRT-PCR, design pri