Abhrajit Dutta

XII-B
Holiday Homework for Biology

THE RT-
PCR TEST
FOR
COVID-19
An in-depth look into the medical test
that saves lives in current times
O
B  Understanding what RT-
PCR is

J  Reviewing the uses of

RT-PCR
E  Analyzing the working of
C RT-PCR

T  Viewing into the various

Methods of RT-PCR

I  Studying the quantitation

of RT-PCR results
V
 Acquiring knowledge of
E RT-PCR instrumentation

S
WHAT IT IS
• RT-PCR stands for Reverse Transcription
Polymerase Chain Reaction.

• The COVID-19 RT-PCR Test is a real-time

test for the qualitative detection of
nucleic acid from SARS-CoV-2
(Coronavirus) in the respiratory
specimens (nasal swabs, sputum,
aspirates, bronchoalveolar lavage etc.)

• Real-Time PCR allows us to measure

minute amounts of DNA sequences in a
sample.

Originally, the method used

radioactive isotope markers to
detect targeted genetic materials,
but subsequent refining has led to
the replacement of isotopic
labelling with special markers,
most frequently fluorescent dyes.
This technique allows scientists to
see the results almost immediately
while the process is still ongoing,
whereas conventional RT–PCR
only provides results at the end of
the process.
USES OF RT-PCR
• Gene expression analysis
 Cancer research
 Drug research

• Disease diagnosis and management

 Viral quantification

• Food testing
 Percent GMO food

• Animal and plant breeding

 Gene copy number

Our objective in this project is to highlight the use of RT-

PCR in Disease Diagnosis and Management

Virus
DISEASE MANAGEMENT
DNA
RNA
BRCA1
mRNA

Seed
wt DNA
Protein
GMO DNA
GENE EXPRESSION ANALYSIS FOOD TESTING
HOW RT-PCR WORKS
• RT-PCR uses RNA as starting material for in
vitro nucleic acid amplification.

• The retroviral Reverse transcriptase is

an RNA-dependent DNA polymerase,
catalyzing DNA synthesis using RNA as the
template.

• The RNA template is converted into

complementary DNA (cDNA) by reverse
transcriptase.

• The cDNA serves later as a template for

exponential amplification using PCR.
CONTINUED…
At the start of a PCR reaction, reagents are in
excess, template and product are at low enough
concentrations that product renaturation does
not compete with primer binding, and
amplification proceeds at a constant,
exponential rate.

The point at which the reaction rate ceases to

be exponential and enters a linear phase of
amplification is extremely variable, even among
replicate samples, but it appears to be primarily
due to product renaturation competing with
primer binding (since adding more reagents or
enzyme has little effect).

At some later cycle the amplification rate drops

to near zero (plateaus), and little more product
is made.

Real-time PCR quantitates reaction products for

each sample in every cycle. The result is an
amazingly broad 107-fold dynamic range, with
no user intervention or replicates required. Data
analysis, including standard curve generation
and copy number calculation, is performed
automatically. With increasing numbers of labs
and core facilities acquiring the instrumentation
required for real-time analysis, this technique is
becoming the dominant RT-PCR-based
quantitation technique.
RT-PCR CHEMISTRIES
Currently four different chemistries, TaqMan,
Molecular Beacons, Scorpions and SYBR Green
(Molecular Probes), are available for real-time PCR.
All of these chemistries allow detection of PCR
products via the generation of a fluorescent signal.
1. TaqMan Probes

TaqMan probes depend on

the 5'- nuclease activity of
the DNA polymerase used
for PCR to hydrolyze an
oligonucleotide that is
hybridized to the target
amplicon. TaqMan probes
are oligonucleotides that have a fluorescent
reporter dye attached to the 5' end and a
quencher moeity coupled to the 3' end. These
probes are designed to hybridize to an internal
region of a PCR product. In the unhybridized
state, the proximity of the fluor and the
quench molecules prevents the detection of
fluorescent signal from the probe. During PCR,
when the polymerase replicates a template on
which a TaqMan probe is bound, the 5'-
nuclease activity of the polymerase cleaves
the probe. This decouples the fluorescent and
quenching dyes and FRET no longer occurs.
Thus, fluorescence increases in each cycle,
proportional to the amount of probe cleavage.
CONTINUED…
Well-designed
TaqMan probes
require very little
optimization. In
addition, they can
be used for
multiplex assays by 2. Molecular Beacons
designing each
probe with a Molecular Beacons also use
spectrally unique FRET to detect and quantitate
fluor/quench pair. the synthesized PCR product
However, TaqMan via a fluor coupled to the 5'
probes can be end and a quench attached to
expensive to the 3' end of an oligonucleotide
synthesize, with a substrate. Unlike TaqMan
separate probe probes, Molecular Beacons are
needed for each designed to remain intact
mRNA target being during the amplification
analyzed. reaction, and must rebind to
target in every cycle for signal
measurement. Molecular
Beacons form a stem-loop
structure when free in solution.
Thus, the close proximity of
the fluor and quench molecules
prevents the probe from
fluorescing. When a Molecular
Beacon hybridizes to a target,
the fluorescent dye and
quencher are separated, FRET
does not occur, and the
fluorescent dye emits light
upon irradiation.
CONTINUED…
3. Scorpions 4. SYBR Green

With Scorpion probes, SYBR Green provides the

sequence-specific priming and simplest and most economical
PCR product detection is format for detecting and
achieved using a single quantitating PCR products in
oligonucleotide. The Scorpion real-time reactions. SYBR Green
probe maintains a stem-loop binds double-stranded DNA, and
configuration in the upon excitation emits light. Thus,
unhybridized state. The as a PCR product accumulates,
fluorophore is attached to the fluorescence increases. The
5' end and is quenched by a advantages of SYBR Green are
moiety coupled to the 3' end. that it is inexpensive, easy to use,
The 3' portion of the stem also and sensitive. The disadvantage
contains sequence that is is that SYBR Green will bind to
complementary to the any double-stranded DNA in the
extension product of the reaction, including primer-
primer. This sequence is linked dimers and other non-specific
to the 5' end of a specific reaction products, which results
primer via a non-amplifiable in an overestimation of the target
monomer. After extension of concentration. For single PCR
the Scorpion primer, the product reactions with well
specific probe sequence is able designed primers, SYBR Green
to bind to its complement can work extremely well, with
within the extended amplicon spurious non-specific
thus opening up the hairpin background only showing up in
loop. This prevents the very late cycles.
fluorescence from being
quenched and a signal is
observed.

SYBR Green is the most economical choice for real-time PCR product detection. Since the dye binds to
double-stranded DNA, there is no need to design a probe for any particular target being analyzed.
However, detection by SYBR Green requires extensive optimization. Since the dye cannot distinguish
between specific and non-specific product accumulated during PCR, follow up assays are needed to
validate results.
QUANTITATION OF RT-PCR
RESULTS
1. Standard Curve Method
In this method, a standard curve is first constructed from an RNA
of known concentration. This curve is then used as a reference
standard for extrapolating quantitative information for mRNA
targets of unknown concentrations. Though RNA standards can be
used, their stability can be a source of variability in the final
analyses. In addition, using RNA standards would involve the
construction of cDNA plasmids that have to be in vitro transcribed
into the RNA standards and accurately quantitated, a time-
consuming process. However, the use of absolutely quantitated
RNA standards will help generate absolute copy number data.

In addition to RNA, other nucleic acid samples can be used to

construct the standard curve, including purified plasmid dsDNA, in
vitro generated ssDNA or any cDNA sample expressing the target
gene. Spectrophotometric measurements at 260 nm can be used
to assess the concentration of these DNAs, which can then be
converted to a copy number value based on the molecular weight
of the sample used. cDNA plasmids are the preferred standards for
standard curve quantitation. However, since cDNA plasmids will
not control for variations in the efficiency of the reverse
transcription step, this method will only yield information on
relative changes in mRNA expression. This, and variation
introduced due to variable RNA inputs, can be corrected by
normalization to a housekeeping gene.
CONTINUED…
2. Comparative Ct Method
Another quantitation approach is termed the comparative Ct
method. This involves comparing the Ct values of the samples of
interest with a control or calibrator such as a non-treated sample
or RNA from normal tissue. The Ct values of both the calibrator
and the samples of interest are normalized to an appropriate
endogenous housekeeping gene.

INSTRUMENTATION FOR
REAL-TIME PCR
Real-time PCR requires an instrumentation platform that consists
of a thermal cycler, a computer, optics for fluorescence
excitation and emission collection, and data acquisition and
analysis software. These machines, available from several
manufacturers, differ in sample capacity (some are 96-well
standard format, others process fewer samples or require
specialized glass capillary tubes), method of excitation (some use
lasers, others broad spectrum light sources with tunable filters),
and overall sensitivity. There are also platform-specific
differences in how the software processes data. Real-time PCR
machines are not inexpensive, currently about $25K - $95K, but
are well within purchasing reach of core facilities or labs that
have the need for high throughput quantitative analysis. For a
comprehensive list of real-time thermal cyclers please see the
weblink at the end of this article.
EPILOGUE
Given the number of choices available for every
aspect of real-time PCR, it may be difficult to
determine what detection chemistry, quantitation
method, normalization gene, etc., to use.

Although every experimental situation is unique and

requires specialized consideration, some general
guidelines can be suggested. In terms of quantitation
method (absolute versus relative), the majority of
users will not require absolute data such as copy
number of transcripts or nanograms of DNA, and
therefore, relative quantitation will suffice.

While amplification efficiency may be more

efficiently calculated from raw fluorescence data
instead of a standard curve, using a set of serial
dilutions is recommended not only to check the
dynamic range of the assay but also to ensure the
accuracy of the quantitation.

In addition, inclusion of a standard curve would allow

results to be calculated using any of the relative
quantitation methods available.

Because real-time PCR is now a common method for

measuring gene expression, it is increasingly
important for users to be aware of the numerous
choices available in all aspects of this technology.
Unlike traditional PCR, there are many complexities
with real-time PCR that can affect overall results.
However, with a well-designed experiment performed
with the proper controls, real-time PCR can be one of
the most sensitive, efficient, fast, and reproducible
methods of measuring gene expression.