Beginner's Guide

A beginner’s guide to RT-­PCR,

qPCR and RT-­qPCR
Grace Adams (University The development of the polymerase chain reaction (PCR), for which Kary Mullis received the 1992
of Leicester, UK) Novel Prize in Chemistry, revolutionized molecular biology. At around the time that prize was
awarded, research was being carried out by Russel Higuchi which led to the discovery that PCR can
be monitored using fluorescent probes, facilitating quantitative real-­time PCR (qPCR). In addition,

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the earlier discovery of reverse transcriptase (in 1970) laid the groundwork for the development of
RT-­PCR (used in molecular cloning). The latter can be coupled to qPCR, termed RT-­qPCR, allowing
analysis of gene expression through messenger RNA (mRNA) quantitation. These techniques and their
applications have transformed life science research and clinical diagnosis.

Why are RT-PCR, qPCR and RT-qPCR not gene expression changes (see Figure 1C). Despite these
one and the same? standardized abbreviations, it is important to note that
this nomenclature guideline is not always adhered to,
When discussing this topic, it is important to highlight and qPCR is commonly used to describe RT-­qPCR.
the common misconception that RT-­PCR, qPCR and Similarly, RT is used to denote real-­time PCR rather than
RT-­ qPCR are synonymous. Indeed, the similarities reverse transcription, thus causing confusion over which
between the closely related techniques often result method is being described. For this Beginner’s Guide, we
in the incorrect use of the acronyms. In an attempt to will be using the MIQE abbreviations as described above.
prevent this, the Minimum Information for Publication
of Quantitative Real-­Time PCR Experiments (MIQE) Overview of qPCR and RT-qPCR
guidelines, first published in 2009, proposed a
standardization of abbreviations. They stated that Quantitative PCR, whether involving a reverse
‘RT-­ PCR’ should only be used to describe reverse transcription step or not, is routinely used in molecular
transcription PCR and not real-­time PCR, as is often biology labs and has revolutionized the way in which
confused. Reverse transcription PCR allows the use of research is carried out due to its relatively simple
RNA as a template to generate complementary DNA pipeline (Figure 2). Its advantages over standard PCR
(cDNA). Using the reverse transcriptase enzyme, a include the ability to visualize which reactions have
single-­stranded copy of cDNA is generated. This can worked in real time and without the need for an agarose
then be amplified by a DNA polymerase, generating gel. It also allows truly quantitative analysis. One of
double-­stranded cDNA, feeding into a standard PCR-­ the most common uses of qPCR is determining the
based amplification process (see Figure 1A). This copy number of a DNA sequence of interest. Using
technique can be used in molecular cloning of genes absolute quantitation, the user is able to determine the
of interest (GOIs), but most commonly, it serves as the target copy numbers in reference to a standard curve of
first step in RT-­qPCR. According to MIQE, the acronym defined concentration in a far more accurate way than
‘qPCR’ describes quantitative real-­time PCR, which is ever before. RT-­qPCR, on the other hand, allows the
the PCR amplification of DNA in real time, measured by investigation of gene expression changes upon treatment
a fluorescent probe, most commonly an intercalating dye of model systems with inhibitors, stimulants, small
or a hydrolysis-­based probe, enabling quantitation of the interfering RNAs (siRNAs) or knockout models, etc.
PCR product (see Figure 1B). This technique is used to This technique is also routinely used to detect changes
detect the presence of pathogens and to determine the in expression both prior to (as quality control) and after
copy number of DNA sequences of interest. The final (confirmation of change) RNA-­Seq experiments.
acronym ‘RT-­qPCR’ is used for reverse transcription
quantitative real-­ time PCR. This is a technique Sample preparation
which combines RT-­ PCR with qPCR to enable the The most crucial step in the qPCR and RT-­qPCR pipeline
measurement of RNA levels through the use of cDNA is arguably sample isolation. No matter how good your
in a qPCR reaction, thus allowing rapid detection of assay design is, if the starting material is contaminated

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 Beginner's Guide

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Figure 1. Schematic comparing RT-­PCR, qPCR and RT-­qPCR. (A) RT-­PCR workflow. RNA is isolated and cDNA is generated
via reverse transcription (RT); PCR is then carried out to amplify areas of interest. (B) qPCR schematic. DNA is isolated and
amplified; amplification is quantitated using a probe which fluoresces upon intercalation with double-­stranded DNA. (C) RT-­
qPCR procedure. RNA is isolated and cDNA generated before commencing a qPCR procedure.

or degraded, you will not get accurate results. A good-­ the precursor of protein synthesis), transfer RNA (tRNA;
quality sample is the starting block of good-­quality data. decodes mRNA during translation with the ribosome)
When isolating DNA for qPCR, it is essential that it is and ribosomal RNA (rRNA; reads the amino acid order
free from contaminants that may inhibit the reaction. during translation and links them with the ribosome),
Most often, extraction is carried out using commercially but often (not always) fails to isolate smaller RNAs
available kits, which have the advantage of being user-­ such as non-­ coding RNA (ncRNA; functional RNA
friendly, simple and quick, especially when integrated transcribed from DNA, but not translated into proteins)
with a robotic system. The type of RNA extraction and micro RNAs (miRNAs; regulate gene expression
carried out depends on the type of RNA required. The by inhibiting mRNA translation). With the explosion
most common extraction method used is with total RNA of interest in enhancer RNAs (eRNAs; small RNAs
extraction kits. This isolates messenger RNA (mRNA; transcribed from enhancers) which can vary in length

Figure 2. Workflow of a standard qPCR and RT-­qPCR experiment. Following sample isolation, the integrity is analysed
prior to cDNA generation and commencement of the qPCR assay using either intercalating dyes or hydrolysis probes.
Fluorescence is detected throughout the PCR cycles and used to generate an amplification curve which is used to quantitate
the target sample during data analysis.

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Beginner's Guide
considerably, it is essential that the extraction methods experiment (termed one-­step RT-­qPCR) or is generated
are carefully considered to ensure isolation of the RNA separately from the qPCR (two-­ step RT-­qPCR), as
of interest. In addition to extraction considerations, it is shown in Figure 3. The advantage of one-­step RT-­qPCR
essential that RNA is not contaminated with DNA, since is that there is less experimental variation and fewer risks
this cannot be distinguished from cDNA in the qPCR of contamination, as well as enabling high-­throughput
reaction. To overcome this, most protocols rely on the screening; hence, this option is usually used for clinical
use of a DNase I treatment which digests any DNA. screening. However, it does mean that the sample can
During isolation, sample degradation is always a only be used a limited number of times, whereas two-­
possibility. Accordingly, any good pipeline will involve a step RT-­qPCR enables more reactions per sample and
quality control step to assess the integrity of the sample. flexible priming options and is usually the preferred
This can be done quickly by evaluating the A260/280 ratio option for wide-­scale gene expression analysis, but does
(comparing the absorbance at 260 vs 280 nm, a measure require more optimization.
of contamination by proteins) and the A260/230 ratio

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(260 vs 230 nm, an indication of the presence of organic
contaminants) of the sample; however, this is not very Detection methods
accurate and is subject to interference from several The next most important decision when designing your
factors. A more accurate measure is the use of a virtual experimental pipeline is choosing the method of detection.
gel electrophoresis system such as the Aligent Bioanalyser. All are based on the emission of fluorescence, but the
This system works using a chip that separates RNA based chemistry behind them differs. One method is the use of
on size and detects RNA by fluorescent dyes. This is then a fluorescent dye which binds non-­specifically to double-­
translated to a computer which, using an algorithm, stranded DNA as it is generated. SYBR® Green is the
produces an RNA integrity number (RIN) which represents most common intercalating dye and emits a fluorescent
the quality of the sample, with 10 being the highest. signal upon intercalating with newly synthesized DNA.
The final step in sample preparation for RT-­qPCR The more the DNA generated in the qPCR reaction, the
is the generation of cDNA. cDNA utilizes RT-­ PCR more fluorescence is detected (Figure 4A). The second
(Figure 1) to generate cDNA from the RNA template method of detection uses hydrolysis probes such as
using a reverse transcriptase. This can be done employing TaqMan® probes, which depend on Förster Resonance
oligo(dT) primers, which anneal to the polyA tail of Energy Transfer (FRET) preventing the dye moiety from
RNA, or using random hexamers (primers of six to nine emitting a signal via the quencher when the probe is intact
bases long, which anneal at multiple points along the (Figure 4B). These probes are specific sequences which are
RNA transcript). Generally, a mix of the two primers is designed to bind downstream of the qPCR primers. The
thought to be best as it enables amplification of polyA 5′ end of the probe is labelled with a fluorescent reporter
tail RNA (mainly mRNA) and non-­polyA–containing such as the carboxyfluorescein (FAM) moiety; on the 3′
RNA (tRNA, rRNA, etc). In addition to the primer end is a quencher molecule which prevents fluorescent
consideration, cDNA generation can be part of the qPCR emission when in close proximity to the reporter. As

Figure 3. One-­step vs two-­step RT-­qPCR. One-­step RT-­qPCR involves the generation of cDNA via reverse transcription and
qPCR amplification of the target sequence in one reaction. Two-­step RT-­qPCR separates out the two steps (RT-­PCR and qPCR),
thus enabling more target sequences to be analysed in the qPCR reaction.

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 Beginner's Guide

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Figure 4. Comparison of intercalating dye and hydrolysis-­based probe detection. (A) SYBR® Green detection: Following
denaturation of cDNA, primers anneal and are extended. During extension, SYBR Green binds to the double-­stranded DNA
(dsDNA), emitting a fluorescent signal detected by the qPCR instrument.(B) TaqMan® probe detection: TaqMan probes
bind downstream of the primers to single-­stranded cDNA. During extension, the polymerase breaks up the probe, allowing
the fluorescent signal to be detected due to the loss of proximity to the quencher moiety. (C) Melt curve graph for primer
specificity: A melt curve measures the dissociation of dsDNA at high temperatures. A single DNA species (produced from a
specific primer pair) will result in a single peak (black line), and multiple DNA species or primer dimers will result in two or
more peaks (purple) and indicate non-­specific primers.

DNA polymerase extends the primer, the probe is cleaved, This can also be aided by the use of carefully designed
enabling the reporter molecule to emit a fluorescent signal. primers and by validating their specificity, for which
Since such probes are target specific, they inherently there are many examples online including the
have greater specificity than intercalating dyes. Harvard primer bank. More recently, new-­generation
Consequently, when you detect a signal using a probe, intercalating dyes such as EvaGreen® have been
you can be confident that the signal is genuinely from developed, which have lower background noise and a
your GOI, since it requires the primers and the probe stronger signal, enabling improved melt curve analysis
to bind at the target sequence for signal detection. and amplification detection. Despite their disadvantages,
Intercalating dyes, however, are non-­ specific, and intercalating dyes are significantly cheaper to use than
therefore, further downstream analysis in the form of probes, as you can use the same dye for multiple different
a melt curve is required to ensure that the signal being primer pairs (as long as the reactions are run separately).
detected is genuinely the target of interest (Figure 4C). Since hydrolysis probes are sequence specific, every GOI

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Beginner's Guide
requires an individual set of primer pairs and probe. In of fluorescence. As the reaction progresses into the
consequence, this method is usually only chosen if the exponential growth, fluorescence reaches a level which is
user wants to measure just a few targets of interest, such significantly higher than the baseline; this is referred to
as in diagnostic testing. Since the development of the first as the threshold level. The threshold level is the heart of
commercial qPCR machines, instrumentation has come quantitation, as the point at which your sample crosses
a long way in terms of both reliability and sensitivity. this threshold is recorded as the Ct or Cq value. The
From the first machines, which could measure a small threshold is set in the exponential phase, so the reading is
number of samples, we are now able to carry out high-­ not affected by reagent shortages, etc. in the plateau phase.
throughput screening using 96- and 384-­well plates. This The second crucial factor in quantitation is the use of a
advance is further enhanced through the development reference gene (RG), an endogenous control present in
of detection systems. The detection of multiple emission all samples at a consistent concentration which does not
spectra in many newer machines enables multiplexing of change in response to biological conditions. Often, genes
up to five or six colours at one time, facilitating high-­ such as GAPDH and β-actin are used; however, the levels

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throughput analysis in shorter periods of time. of these transcripts can change in certain conditions, thus
it is essential that the RGs are matched to the experiment.
Quantitation and data analysis To analyse the data, there are two types of
Real-­
time detection of the qPCR cycle results in an quantitation methods to choose from, absolute and
amplification curve with initiation, exponential and relative. Absolute quantitation is the most rigorous in
plateau phases (Figure 5A). This curve forms the basis terms of controls. Each reaction requires a standard of
of quantitation. When amplification starts, the level of known concentration for the RG and GOI, for which a
fluorescence is low and is used to set the baseline level standard curve is generated using the log concentrations

Figure 5. Quantitation of RT-­qPCR and qPCR. (A) Amplification curve generated during the run as the reaction is measured
in real time. Due to fluorescence detection, an amplification curve is generated (blue curve) which involves an initiation
phase [low level of fluorescence, often termed the baseline (black line)]. A threshold is determined (green line) once the
amplification curve is in the exponential phase, and where the amplification crosses this line determines the Cq/Ct value used
to quantify data. Negative controls, i.e., water controls, should be around the baseline value. (B) A serial dilution of standards
of known concentration are used to generate a amplification curve, which when Cq values are plotted against their log
concentrations produce a standard curve. Target sequences of unknown concentrations can then be accurately quantified
using their Cq as shown by the hashed cyan line.

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 Beginner's Guide
and the Ct value (Figure 5B). This standard curve can to real-­time RT-­PCR or RT-­qPCR allows high-­throughput
then be used to quantitate the concentration of the screening of patients, which is critical during a public
unknown experimental samples and is often used for health emergency. These tests have been rapidly designed
identifying DNA copy numbers. The second approach following the deposition of the SARS-­ CoV-2 genome
is relative quantitation, which enables you to calculate allowing prompt design of primers and probes specific for
the ratio between the RG and the GOI. The accuracy Covid-19. The most common test for SARS-­CoV-2, which
of this quantitation depends on the RG; therefore, it is has been implemented by the World Health Organization
crucial that this remains unchanged, so as to prevent (WHO), Public health England (PHE) and National
erroneous results. The method used to express the Health Service (NHS) laboratories, is real-­time RT-­PCR
ratio between the RG and GOI is called the delta delta (RT-­qPCR) using a system similar to TaqMan probes. The
Ct method (2-ΔΔCq). The Ct (Figure 5A) of the RG is Drosten group, based in Berlin, has designed a real-­time
removed from the GOI Ct, so as to remove any errors RT-­PCR assay which detects the RdRp gene of SARS-­CoV-2
in sample loading. This generates a ΔCt value for all

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and involves isolation of RNA and subsequent one-­step
samples, which is then compared back to a control real-­time RT-­PCR using fluorescent probes designed for the
sample to generate the ΔΔCt. This method is generally RdRp cDNA. A second collaborative group based in Hong
used for comparing healthy vs disease samples, etc. Kong has designed a similar test employing two one-­step
RT-­qPCR assays using fluorescent probes for alternative
SARS-­CoV-2 genes, called ORF1b and the N gene. These
Covid-19: the new frontier for real-time two real-­time assays can be scaled up onto large automated
PCR assays qPCR machines, thus enabling rapid detection with high
sensitivity and selectivity over similar coronaviruses such
RT-­PCR has been used to detect the viruses responsible for as the virus causing SARS. Consequently, it is clear that
respiratory infections in public health for many years. With as well as being a powerful investigative technique in life
the recent outbreak of SARS-­CoV-2, the virus causing sciences research labs, this technique is a strong contender
Covid-19, the use of real-­time RT-­PCR has come to the for rapid diagnostics in current and future public health
forefront of research. The conversion of RT-­PCR testing emergencies. ■
Further reading

• Liu, Y., Cao, Y., Wang, T. et al. (2019) Detection of 12 common food-­borne bacterial pathogens by TaqMan Real-­Time
PCR using a single set of reaction conditions, Front. Microbiol. 10, 222 DOI: 10.3389/fmicb.2019.00222
• Bustin, S.A. Benes, V., Garson, J.A., et al. (2009) The MIQE guidelines: Minimum Information for publication of
Quantitative Real-­Time PCR Experiments, Clin. Chem. 55, 611–622. DOI: 10.1373/clinchem.2008.112797
• Nolan, T., Huggett, J. and Sanchez, E. (2013) Good practice guide for the application of quantitative PCR (qPCR), LGC .
https://www.gene-quantification.de/national-measurement-system-qpcr-guide.pdf [Accessed 10 June 2020]
• Livak, K.J. and Schmittgen, T.D. (2001) Analysis of relative gene expression data using real-­time quantitative PCR and
the 2(-­Delta Delta C(T)) Method. Methods. 25, 402–408. DOI:10.1006/meth.2001.1262
• Sheridan, C. (2020) Coronavirus and the race to distribute reliable diagnostics, Nat. Biotechnol. 38, 379–391 DOI:
10.1038/d41587-020-00002-2
• Corman, V.M., Landt, O., Kaiser, M., et al. (2020) Detection of 2019 novel coronavirus (2019-­nCoV) by real-­time RT-­PCR.
Euro. Surveill. 25, DOI: 10.2807/1560-7917.ES.2020.25.3.2000045
• Chu, D.K.W., Pan, Y., Cheng, S.M.S., et al. (2020) Molecular diagnosis of a novel coronavirus (2019-­nCoV) causing an
outbreak of pneumonia, Clin. Chem. 66, 549–555 DOI: 10.1093/clinchem/hvaa029

Grace Adams is a postdoctoral researcher in the Department of Molecular and Cell Biology at the University
of Leicester. She started in the field of Biochemistry in 2010 as an undergraduate at the University of Leicester.
During her PhD, she worked with Professor Shaun Cowley to study the role of Class I Histone Deacetylases
in gene expression. In both her PhD and postdoctoral work Grace used RT-­qPCR extensively to study gene
expression changes. Email: [email protected]

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