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Quantitative PCR (qPCR), also known as real-time PCR, has significantly impacted the field of molecular biology by enabling the accurate and quantitative measurement of gene expression levels. This powerful technique combines the amplification capabilities of traditional PCR with real-time detection, allowing researchers to monitor the accumulation of PCR products as they form. It can be used for absolute quantification in addition to relative quantification. qPCR is extensively used in various applications, including gene expression profiling, verification of microarray results, and detection of genetic mutations. One of the key advantages of qPCR is its ability to provide quantitative data that is both sensitive and specific, making it possible to detect even low-abundance transcripts in complex biological samples.

The process of qPCR gene expression analysis involves several important steps, including the extraction of high-quality RNA, reverse transcription to generate complementary DNA (cDNA), and the amplification and detection of target sequences using fluorescent dyes or probes. The choice of appropriate reference genes for normalization is also important for consistent and reliable results. This introduction aims to provide an overview of the fundamental principles of qPCR gene expression analysis, highlighting the key steps, considerations, and best practices that are important for effective implementation of this technique in research settings.

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What is gene expression?

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 biological regulatory functions [1].

When studying gene expression with real-time PCR, scientists commonly investigate changes—increases or decreases—in the expression of a specific 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, or qPCR, is used by numerous scientists performing gene expression.


Reverse transcription quantitative PCR (RT-qPCR)

cDNA reverse transcription icon

qPCR (quantitative PCR)/Real-time PCR: These are synonymous and refer to the same technique of amplifying and quantifying DNA in real time.

RT-qPCR (reverse transcription quantitative PCR): This involves an additional step of reverse transcription of RNA to cDNA before performing qPCR. RT-qPCR refers to the quantification of RNA after reverse transcription into cDNA.

RT-qPCR is one of the most widely used 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 (i.e., a TaqMan probe or SYBR Green dye) to monitor the accumulation of PCR product. As the quantity of target amplicon increases, so does the amount of fluorescence emitted from the fluorophore.

Real-time PCR advantages

Advantages of real-time PCR

  • 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
Traditional PCR end-point phase measurement

End-point phase measurement in traditional PCR

There are three phases in a basic PCR run:

  1. Exponential phase—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. The exponential phase provides the most reliable data for quantification because the reaction efficiency is highest and most consistent during this phase.
  2. Linear (high variability) phase—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.
  3. 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.
Real-time PCR exponential phase measurement

Exponential phase measurement in real-time PCR

RT-qPCR 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.


Selecting the appropriate detection chemistry

The two types of chemistries that have been developed for gene expression studies using real-time PCR are 1) TaqMan chemistry (also known as “fluorogenic 5´ nuclease chemistry”) and 2) SYBR Green dye chemistry.

Learn more about TaqMan vs. SYBR chemistry for real-time PCR


Selecting the reverse transcription method

RT-qPCR can be performed as a one-step or two-step procedure. The commonly used method for studying gene expression is two-step RT-qPCR because it offers flexibility in primer selection and the ability to store cDNA. However, one-step PCR is typically faster and reduces the risk of contamination.

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 beneficial when studying a single gene in many samples.

Learn more: One-step RT-PCR systems

About two-step RT-qPCR

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.

Learn more: One-step vs two-step RT-PCR


RT-qPCR assay design and selection

assay development icon

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.

Target of interest

Identify the gene(s) or pathway of interest.

Specificity

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 .

Efficiency

The recommended amplification efficiency of your assay is between 90–110%. Less efficient assays may result in reduced sensitivity and linear dynamic range, thereby limiting your ability to detect low abundance transcripts.

Reproducibility

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.

Predesigned assays and PCR arrays

Whether you are studying single genes or whole pathways, you now have numerous 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:

Endogenous controls

In any gene expression study, selecting a valid normalization or endogenous control to correct for differences in RNA sampling is important 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.

Learn more: How to select endogenous controls

Singleplex PCR vs duplex PCR vs multiplex PCR

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.

Commonly, 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.

Consider the following advantages and limitations when choosing between duplex, singleplex, and multiplex PCR: 

PCRDescriptionAdvantageLimitation
SingleplexA reaction in which a single target is amplified in the reaction tube or well.
  • No optimization is required for TaqMan assays.
  • Flexibility to use TaqMan or SYBR Green reagents.
Requires separate reactions for the target and the endogenous control assay.
DuplexA reaction in which two targets are amplified in the same reaction tube or well.

Reduces the:

  • Running costs.
  • Dependence on accurate pipetting

Requires optimization.

Potential for primer-dimer formation.

MultiplexA reaction in which multiple DNA target sequences (more than two) are amplified in the same reaction tube or well.
  • Same as duplex PCR
  • Highly efficient for analyzing multiple targets simultaneously

Complex optimization and potential for cross-reactivity.

Difficult to balance amplification efficiency for all targets.

NOTE: We have predesigned assays to select a VIC reporter dye.


Selecting the quantitation method

quality control pcr icon

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 qPCR 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 [4].

Experimental verification

To use the comparative CT method, run a verification experiment to show that the efficiencies of the target and endogenous control amplifications are approximately equal [4].

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.

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 less common method for quantitation of gene expression.

Guidelines for selecting a quantitation method

Consider the following advantages and limitations when selecting the quantitation method.

Experiment typeAdvantageLimitation
Comparative CT (ΔΔCT)
  • Relative levels of target in samples can be determined without the use of a standard curve or dilution series.
  • Requires reduced reagent usage.
  • More space is available in the reaction plate.
  • Because a standard curve is not needed, throughput can increase.
  • Dilution errors made in creating the standard curve samples are eliminated.
  • The target and endogenous control can be amplified in the same tube, increasing throughput and reducing pipetting errors.
  • Suboptimal (low PCR efficiency) assays may produce inaccurate results.
  • Before you can use the comparative CT method, the PCR efficiencies for the target assay and the endogenous control assay must be approximately equal.
Relative standard curveRequires the least amount of verification 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 qPCR data

analyze icon

For detailed information about data analysis, see the appropriate documentation for your instrument. Use the absolute or relative quantification (ΔΔCT) methods to analyze results.

The general guidelines for analysis include:

  • View the amplification plot; then, if needed:
    • Adjust the baseline and threshold values.
    • Remove outliers from the analysis.
  • In the well table or results table, view the CT values for each well and for each replicate group.

Perform additional analysis using any of the following software:

Find real-time PCR software downloads

Experiment typeAdvantageLimitation
ComparativeCT (ΔΔCT)
  • Relative levels of target in samples can be determined without the use of a standard curve or dilution series.
  • Requires reduced reagent usage.
  • More space is available in the reaction plate.
  • Because a standard curve is not needed, throughput can increase.
  • Dilution errors made in creating the standard curve samples are eliminated.
  • The target and endogenous control can be amplified in the same tube, increasing throughput and reducing pipetting errors.
  • Suboptimal (low PCR efficiency) assays may produce inaccurate results.
  • Before you can use the comparative CT method, the PCR efficiencies for the target assay and the endogenous control assay must be approximately equal.
Relative standard curveRequires 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.

 


References
  1. 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. 
  2. 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. 
  3. Förster, V. T. 1948. Zwischenmolekulare Energiewanderung und Fluoreszenz. Annals of Physics (Leipzig) 2:55–75. 
  4. Livak, K.J. and Schmittgen, T.D. 2008. Analyzing real-time PCR data by the comparative CT method. Nature Protocols 3, 1101–1108. 


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Download PDF:  Getting Started Guide: Introduction to Gene Expression

 

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