Gene expression or messenger RNA (mRNA) analysis is the most commonly-used application for qPCR. Researchers reverse transcribe RNA, then use the cDNA produced as a template in qPCR reactions to detect and quantitate gene expression products
Gene expression is the process by which information from a gene is used in the synthesis of a functional gene product. These products are often proteins, but in nonprotein coding genes such as rRNA genes or tRNA genes, the product is a structural or housekeeping RNA. In addition, small non-coding RNAs (miRNAs, piRNA) and various classes of long non-coding RNAs are involved in a variety of regulatory functions (Taft, R.; Pang, K.C.; Mercer, T.R.; Dinger, M.; and Mattick, J.S. 2010).
When studying gene expression with real-time polymerase chain reaction (PCR), scientists usually investigate changes – increases or decreases – in the expression of a particular gene or set of genes by measuring the abundance of the gene-specific transcript. The investigation monitors the response of a gene to treatment with a compound or drug of interest, under a defined set of conditions. Gene expression studies can also involve looking at profiles or patterns of expression of several genes. Whether quantitating changes in expression levels or looking at overall patterns of expression, real-time PCR is used by most scientists performing gene expression.
Real-time PCR —also known as quantitative reverse transcription PCR (RT-qPCR), and quantitative PCR (qPCR)—is one of the most powerful and sensitive gene analysis techniques available. It is used for a broad range of applications including quantitative gene expression analysis, genotyping, copy number, drug target validation, biomarker discovery, pathogen detection, and measuring RNA interference.
Real-time PCR measures PCR amplification as it occurs, so that it is possible to determine the starting concentration of nucleic acid. In traditional PCR, which is based on end-point detection, results are collected after the reaction is complete, making it impossible to determine the starting concentration of nucleic acid.
Every real-time PCR contains a fluorescent reporter molecule—a TaqMan® probe or SYBR® Green dye, for example—to monitor the accumulation of PCR product. As the quantity of target amplicon increases, so does the amount of fluorescence emitted from the fluorophore.
Advantages of real-time PCR include:
- Generation of accurate quantitative data
- Increased dynamic range of detection
- Elimination of post-PCR processing
- Detection down to one copy
- Increased precision to detect smaller fold changes
- Increased throughput
End-point phase measurement in traditional PCR
There are three phases in a basic PCR run:
Exponential – Exact doubling of product occurs at every cycle (assuming 100% reaction efficiency). Exponential amplification occurs because all of the reagents are fresh and available, the kinetics of the reaction push the reaction to favor doubling of amplicon.
Linear (High Variability) – As the reaction progresses, some of the reagents are consumed as a result of amplification. The reactions start to slow down and the PCR product is no longer doubled at each cycle.
Plateau (End-Point: Gel detection for traditional methods) – The reaction has stopped, no more products are made, and if left long enough, the PCR products begin to degrade. Each tube or reaction plateaus at a different point, due to the different reaction kinetics for each sample. These differences can be seen in the plateau phase. The plateau phase is the end point, where traditional PCR takes its measurement.
Exponential phase measurement in real-Time PCR
Real-Time PCR focuses on the exponential phase, which provides the most precise and accurate data for quantitation. During the exponential phase, the real-time PCR instrument calculates two values:
- Threshold – The level of detection at which a reaction reaches a fluorescent intensity above background.
- CT– The PCR cycle at which the sample reaches the threshold. The CT value is used in absolute or relative quantitation.
- Decide the type of real-time chemistry to use (TaqMan® or SYBR®).
- Select a reverse transcription method.
- Select or design assays.
- Select a quantitation method.
The workflow for performing gene expression using real-time PCR is shown:
The following topic, “Selecting the detection chemistry”, provides a brief overview of the two real-time PCR chemistries, TaqMan probe and SYBR Green I dye, and guidelines for selecting a chemistry.
The two types of chemistries that have been developed for gene expression studies using real-time PCR are:
- TaqMan chemistry (also known as “fluorogenic 5´ nuclease chemistry”)
- SYBR Green I dye chemistry
About TaqMan chemistry
Real-time PCR systems were improved by the introduction of fluorogenic-labeled probes that use the 5′ nuclease activity of Taq DNA polymerase. The availability of these fluorogenic probes enabled the development of a real-time method for detecting only specific amplification products.
About TaqMan probes
TaqMan probes are dual labeled, hydrolysis probes that increase the specificity of real-time PCR assays. TaqMan probes contain:
- A reporter dye (for example, FAM™ dye) linked to the 5′ end of the probe
- A nonfluorescent quencher (NFQ) at the 3′ end of the probe
- MGB moiety attached to the NFQ
TaqMan MGB probes also contain a minor groove binder (MGB) at the 3´ end of the probe. MGBs increase the melting temperature (Tm) without increasing probe length ; allowing for the design of shorter probes (Afonina et al., 1997; Kutyavin et al., 1997).
How TaqMan real-time chemistry works
Here is how TaqMan real-time chemistry works:
- An oligonucleotide probe is constructed with a fluorescent reporter dye bound to the 5′ end and a quencher on the 3′ end. While the probe is intact, the proximity of the quencher greatly reduces the fluorescence emitted by the reporter dye by fluorescence resonance energy transfer through space (Förster, V. T. 1948).
- If the target sequence is present, the probe anneals between primer sites and is cleaved by the 5′ nuclease activity of the taq DNA polymerase during extension.
- Separates the reporter dye from the quencher, increasing the reporter dye signal.
- Removes the probe from the target strand, allowing primer extension to continue to the end of the template strand. Thus, inclusion of the probe does not inhibit the overall PCR process
- Additional reporter dye molecules are cleaved from their respective probes with each cycle, resulting in an increase in fluorescence intensity proportional to the amount of amplicon produced. The higher the starting copy number of the nucleic acid target, the sooner a significant increase in fluorescence is observed.
About SYBR Green I dye chemistry
SYBR Green I dye chemistry uses SYBR Green I dye, which binds to double-stranded DNA, to detect PCR products as they accumulate during PCR cycles.
An important difference between the TaqMan probes and SYBR Green I dye chemistries is that the SYBR Green I dye chemistry binds all double-stranded DNA, including nonspecific reaction products. A well-optimized reaction is therefore essential for accurate results.
How SYBR Green I dye chemistry works
The SYBR Green I dye chemistry uses the SYBR Green I dye to detect PCR products by binding to the double-stranded DNA formed during PCR. Here is how this chemistry works:
- SYBR Green I dye fluoresces when bound to double-stranded DNA.
- During the PCR, taq DNA Polymerase amplifies the target sequence, which creates the PCR product, or “amplicon.”
- As the PCR progresses, more amplicons are created. Since SYBR Green binds to all double-stranded DNA, the result is an increase in fluorescent intensity proportional to the amount of PCR product produced.
Comparison of TaqMan and SYBR Green I Dye chemistries
The illustration below shows a comparison of TaqMan-based and SYBR Greenbased detection workflows.
Comparison of TaqMan and SYBR Green chemistries
TaqMan probe-based chemistry and SYBR Green I dye can be used for the assay types listed below.
|SYBR Green I Dye||Yes||No|
Each chemistry has its advantages and limitations. For example, TaqMan chemistry enables you to perform multiplex PCR. If high sensitivity is your priority, SYBR Green chemistry offers that advantage. Consider the following aspects of each chemistry type when choosing between TaqMan probe-based and SYBR Green chemistry for your assays:
|TaqMan||Uses a fluorogenic probe to enable the detection of a specific PCR product as it accumulates during PCR cycles. Detects specific amplification products only.||A different probe has to be synthesized for each unique target sequence|
|SYBR Green I dye|
Uses SYBR Green I dye, a doublestranded DNA binding dye, to detect PCR product as it accumulates during PCR cycles.
Detects all doublestranded DNA, including both specific and nonspecific reaction products.
Selecting the reverse transcription method
Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) is used to quantify RNA. RT-qPCR can be performed as a one-step or two-step procedure. The most common method for looking at gene expression is two-step RT-qPCR.
About one-step RT-qPCR
With one-step RT-qPCR, the reverse transcription and PCR amplification steps are performed in a single buffer system:
The reaction proceeds without the addition of reagents between the RT and PCR steps. One-step RT-qPCR offers the convenience of a single-tube preparation for RT and PCR amplification. This method is target- or gene-specific. Only your specific target is transcribed because you use one of your PCR primers to prime the reverse transcription. This approach is useful when studying a single gene in many samples.
About two-step RT-PCR
With two-step RT-qPCR, the reverse transcription and PCR amplification steps are performed in two separate reactions:
Two-step RT-qPCR is useful when detecting multiple transcripts from a single sample, or when storing a portion of the cDNA for later use. In a two-step approach, the\ reverse transcription is usually primed with either oligo d(T)16 or random primers. Oligo d(T)16 binds to the poly-A tail of mRNA, and random primers bind across the length of the RNA being transcribed.
Guidelines for selecting the reverse transcription and amplification reagents
For guidelines on selecting the reverse transcription and amplification reagents, see the Applied Biosystems Gene Expression Assays Protocol (PN 4333458) or the Real-Time PCR Decision Tree.
If you have difficult samples, use TaqMan PreAmp Master Mix, which preamplifies small amounts of cDNA without introducing amplification bias to the sample. Preamplification enables you to stretch your limited sample into many more real-time PCR reactions. For more information, see the Applied Biosystems TaqMan PreAmp Master Mix Kit Protocol (PN 4384557A).
When you are deciding whether to select a predesigned assay or design a custom assay, think about your goals for the assay. These considerations should be taken into account whether you purchase commercially available, preformulated primer or probe sets or you design your own assays.
Considerations for optimal assay performance
Target of interest
Identify the gene(s) or pathway of interest.
Depending on the level of specificity you require, you can select or design an assay to:
- Detect all known transcripts of your gene of interest (gene-specific detection).
- Detect a unique splice variant (transcript-specific detection).
- Discriminate between closely related members of a gene family (homologs and potentially orthologs).
Ensure specificity by checking against known sequences databases such as NCBI and Ensembl.
Ensure that your assay has an amplification efficiency close to 100%. Less efficient assays may result in reduced sensitivity and linear dynamic range, thereby limiting your ability to detect low abundance transcripts.
You should be able to repeat your experiment and produce the same results. Factors that could affect reproducibility are oligo manufacturing and assay formulation, and primer dimer formation.
Preformulated assays and PCR arrays
Whether you are studying single genes or whole pathways, you now have many choices of preformulated assays and PCR arrays from multiple vendors:
- Preformulated assays in tubes are appropriate when you are a studying small number of genes or when you need maximum flexibility.
- PCR arrays are 96 or 384 well plates or microfluidic cards loaded with assays corresponding to pathways or other common gene sets. This format is appropriate when you are studying a large number of genes or when you are trying to narrow down the number of genes you want to focus on in your experiment.
If your gene targets are available as commercial primer or probe sets, you could use either of the following tools to design customized assays:
- A public primer or probe design tool, or
- A commercial assay design tool or service, such as the Applied Biosystems Custom TaqMan Assay Design Tool (www.appliedbiosystems.com/tools/cadt/)
In any gene expression study, selecting a valid normalization or endogenous control to correct for differences in RNA sampling is critical to avoid misinterpretation of results. TaqMan Endogenous Controls consist of the most commonly used housekeeping genes in human, mouse, and rat, and these controls are provided as a preformulated set of predesigned probe and amplification primers.
For more information on selecting an endogenous control, refer to Using TaqMan® Endogenous Control Assays to Select an Endogenous Control for Experimental Studies (Publication 127AP08-01)
Singleplex PCR vs. duplex PCR
Duplex PCR is the simultaneous amplification of two target sequences in a single reaction.
Duplex real-time PCR is possible using TaqMan probe–based assays, in which each assay has a specific probe labeled with a unique fluorescent dye, resulting in different observed colors for each assay. Real-time PCR instruments can discriminate between the different dyes. The signal from each dye is used to separately quantitate the amount of each target.
Typically one probe is used to detect the target gene; another probe is used to detect an endogenous control (reference gene). Running both assays in a single tube reduces both the running costs and the dependence on accurate pipetting when splitting a sample into two separate tubes. Duplex PCR is not possible when using SYBR Green chemistry.
Consider the following advantages and limitations when choosing between duplex and singleplex PCR:
|Singleplex||A reaction in which a single target is amplified in the reaction tube or well.||Requires separate reactions for the target and the endogenous control assay.|
|Duplex||A reaction in which two targets are amplified in the same reaction tube or wel|
|Requires validation and optimizatio|
Note: Life Technology has predesigned assays to select a VIC reporter dye.
Methods for relative quantitation of gene expression enable you to quantify differences in the expression level of a specific target (gene) between different samples. The data output is expressed as a fold-change or a fold-difference of expression levels. For example, you might want to look at the change in expression of a particular gene over a given time period in treated versus untreated samples.
When you are designing your Real-Time PCR gene expression experiment, select the method to use to quantify the target sequence:
- Comparative CT (ΔΔCT) method (relative quantitation)
- Relative standard curve method (relative quantitation)
- Standard curve method (absolute quantitation)
Comparative CT method
Relative quantitation is a technique used to analyze changes in gene expression in a given sample relative to a reference sample (such as an untreated control sample).
Comparative CT experiments are commonly used to:
- Compare expression levels of a gene in different tissues.
- Compare expression levels of a gene in treated versus untreated samples.
- Compare expression levels of genes in samples treated with a compound under different experimental conditions, over a time-course-study-defined period of time.
For more information, read Analysis of relative gene expression data using real-time quantitative PCR and the 2-ΔΔ CT method by Kenneth J. Livak and Thomas D. Schmittgen.
To use the comparative CT method, run a validation experiment to show that the efficiencies of the target and endogenous control amplifications are approximately equal (Livak, K.J. and Schmittgen, T.D. 2001).
Relative standard curve method
Similar to the comparative CT method, the relative standard curve method can be used to determine fold changes in gene expression. Generally, use the relative standard curve method when you use two assays for quantitation (an assay for the target gene and an assay for endogenous control) that did not have equivalent amplification efficiency. A dilution series is created from a common sample and run with both the target and the endogenous control gene. For all experimental samples, a quantity is determined from this dilution series, and a fold change in expression can be calculated from this data.
For more information, read the Applied Biosystems User Bulletin for the ABI PRISM 7700 Sequence Detection System: Relative Quantitation of Gene Expression (PN 4303859).
Standard curve method
Use the standard curve method to determine the absolute target quantity in samples. With the standard curve method, the real-time PCR system software measures amplification of the target in samples and in a standard dilution series of known copy number. Data from the standard dilution series are used to generate the standard curve. Using the standard curve, the software interpolates the absolute quantity of target in the samples. The standard curve method is probably the least common method for quantitation of gene expression.
Guidelines for selecting the quantitation method
Consider the following advantages and limitations when selecting the quantitation method.
|Relative standard curve||Requires the least amount of validation because the PCR efficiencies of the target and endogenous control do not need to be equivalent.||A dilution series must be run for each target; a series requires more reagents and more space in the reaction plate.|
|Absolute quantitation(standard curve)||Absolute, rather than relative, quantities of transcripts are calculated.||The required standard curve for each target requires more reagents and more space in the reaction plate.|
Analyzing the data requires you to:
- View the amplification plots for the entire plate.
- Set the baseline and threshold values.
- Use the relative standard curve or the comparative CT method to analyze your data.
Resources for data analysis
The details of data analysis depend on the real-time PCR instrument that you use; refer to the appropriate user guide for instructions on how to analyze your data.
|Real-time PCR system||Document||Part Number|
|7900HT Fast system||Relative Quantitation Using Comparative CT Getting Started Guide||4364016|
|Performing Fast Gene Quantification: Quick Reference Card||4351892|
|Performing Fast Gene Quantitation with 384-Well Plates: User Bulletin||4369584|
|7300/7500/7500 Fastsystem||Relative Quantification: Getting Started Guide||4347824|
|Relative Standard Curve and Comparative CT Experiments GettingStarted Guide||4387783|
|StepOne™/StepOnePlus™system||Comparative CT/Relative Standard Curve and Comparative CT Experiments Getting Started Guide||4376785|
|All||Applied Biosystems 7900HT Fast Real-Time PCR Systems and 7300/7500/7500 Fast Real-Time PCR Systems Chemistry Guide||4348358|
Tools for data analysis
Applied Biosystems recommends the following software for analyzing data generated using TaqMan Gene Expression Assays.
DataAssist™ Software is a simple, yet powerful data analysis tool for sample comparison when using the comparative CT (ΔΔCT) method for calculating relative quantitation of gene expression. The software is compatible with all Applied Biosystems instruments. It contains a filtering procedure for outlier removal, and various normalization methods based on single or multiple genes, and it provides relative quantification analysis of gene expression through a combination of statistical analysis and interactive visualization.
DataAssist™ Software is free and can be downloaded from: www.lifetechnologies.com/dataassist
For more information about comparative CT, read Analyzing real-time PCR data by the comparative CT method by Kenneth J. Livak and Thomas D. Schmittgen.
Real-Time StatMiner® Software from Integromics is a software analysis package for qPCR experiments that is compatible with all Applied Biosystems instruments. Real-Time StatMiner® Software uses a step-by-step analysis workflow guide that includes parametric, non-parametric, and paired tests for relative quantification of gene expression, as well as two-way ANOVA for two-factor differential expression analysis.
- Afonina, I., Zivarts, M., Kutyavin, I., et al. 1997. Efficient priming of PCR with short oligonucleotides conjugated to a minor groove binder. Nucleic Acids Res. 25:2657–2660.
- Förster, V. T. 1948. Zwischenmolekulare Energiewanderung und Fluoreszenz. Annals of Physics (Leipzig) 2:55–75.
- Kutyavin, I.V., Lukhtanov, E.A., Gamper, H.B., and Meyer, R.B. 1997. Oligonucleotides with conjugated dihydropyrroloindole tripeptides: base composition and backbone effects on hybridization. Nucleic Acids Res. 25:3718–3723.
- Lakowicz, J.R. 1983. Energy Transfer. In Principles of Fluorescence Spectroscopy, New York: Plenum Press 303–339.
- Livak, K.J. and Schmittgen, T.D. 2008. Analyzing real-time PCR data by the comparative CT method. Nature Protocols 3, 1101-1108.
- Livak, K.J. and Schmittgen, T.D. 2001. Analysis of relative gene expression data using real-time quantitative PCR and the 2-ΔΔ CT Method. Methods 25, 402–408.
- Livak KJ, Flood SJ, Marmaro J, Giusti W, and Deetz K. 1995. Oligonucleotides with fluorescent dyes at opposite ends provide a quenched probe useful for detecting PCR product and nucleic acid hybridization. PCR Methods Appl 4:357–362.
- Longo, M.C., Berninger, M.S., and Hartley, J.L. 1990. Use of uracil DNA glycosylase to control carryover contamination in polymerase chain reactions. Gene 93:125–128.
- Taft, R.; Pang, K.C.; Mercer, T.R.; Dinger, M.; and Mattick, J.S. 2010. Non-coding RNAs: regulators of disease. Journal of Pathology, 220:126-139