Reverse

transcription
polymerase chain
reaction

Reverse transcription polymerase chain reaction (RT-PCR) is a laborat ory t echnique

combining reverse t ranscript ion of RNA int o DNA (in t his cont ext called complement ary DNA
or cDNA) and amplificat ion of specific DNA t arget s using polymerase chain react ion (PCR).[1]
It is primarily used t o measure t he amount of a specific RNA. This is achieved by monit oring
t he amplificat ion react ion using fluorescence, a t echnique called real-t ime PCR or
quant it at ive PCR (qPCR). Combined RT-PCR and qPCR are rout inely used for analysis of gene
expression and quant ificat ion of viral RNA in research and clinical set t ings.
RT-PCR

The close associat ion bet ween RT-PCR and qPCR has led t o met onymic use of t he t erm
qPCR t o mean RT-PCR. Such use may be confusing,[2] as RT-PCR can be used wit hout qPCR,
for example t o enable molecular cloning, sequencing or simple det ect ion of RNA. Conversely,
qPCR may be used wit hout RT-PCR, for example t o quant ify t he copy number of a specific
piece of DNA.

Nomenclature

The combined RT-PCR and qPCR t echnique has been described as quant it at ive RT-PCR[3] or
real-t ime RT-PCR[4] (somet imes even called quant it at ive real-t ime RT-PCR[5]), has been
variously abbreviat ed as qRT-PCR,[6] RT-qPCR,[7] RRT-PCR,[8] and rRT-PCR.[9] In order t o avoid
confusion, t he following abbreviat ions will be used consist ent ly t hroughout t his art icle:

Technique Abbreviation

Polymerase chain react ion PCR

Reverse t ranscript ion polymerase chain react ion RT-PCR

Real-t ime polymerase chain react ion qPCR

RT-PCR / qPCR combined t echnique qRT-PCR

Not all aut hors, especially earlier ones, use t his convent ion and t he reader should be caut ious
when following links. RT-PCR has been used t o indicat e bot h real-t ime PCR (qPCR) and
reverse t ranscript ion PCR (RT-PCR).

History

Since it s int roduct ion in 1977, Nort hern blot has been used ext ensively for RNA quant ificat ion
despit e it s short comings: (a) t ime-consuming t echnique, (b) requires a large quant it y of RNA
for det ect ion, and (c) quant it at ively inaccurat e in t he low abundance of RNA cont ent .[10][11]
However, since it s invent ion by Kary Mullis in 1983 RT PCR has since displaced nort hern blot
as t he met hod of choice for RNA det ect ion and quant ificat ion.[12]

RT-PCR has risen t o become t he benchmark t echnology for t he det ect ion and/or comparison
of RNA levels for several reasons: (a) it does not require post PCR processing, (b) a wide
range (>107-fold) of RNA abundance can be measured, and (c) it provides insight int o bot h
qualit at ive and quant it at ive dat a.[5] Due t o it s simplicit y, specificit y and sensit ivit y, RT-PCR is
used in a wide range of applicat ions from experiment s as simple as quant ificat ion of yeast
cells in wine t o more complex uses as diagnost ic t ools for det ect ing infect ious agent s such
as t he avian flu virus and SARS-CoV-2.[13][14][15]

Principles

In RT-PCR, t he RNA t emplat e is first convert ed int o a complement ary DNA (cDNA) using a
reverse t ranscript ase (RT). The cDNA is t hen used as a t emplat e for exponent ial amplificat ion
using PCR. The use of RT-PCR for t he det ect ion of RNA t ranscript has revolut ionized t he
st udy of gene expression in t he following import ant ways:

Made it t heoret ically possible t o det ect t he t ranscript s of pract ically any gene [16]
 Enabled sample amplificat ion and eliminat ed t he need for abundant st art ing mat erial
required when using nort hern blot analysis[17][18]

Provided t olerance for RNA degradat ion as long as t he RNA spanning t he primer is int act [17]

One-step RT-PCR vs two-step RT-PCR

One-step vs two-step RT-PCR

The quant ificat ion of mRNA using RT-PCR can be achieved as eit her a one-st ep or a t wo-st ep
react ion. The difference bet ween t he t wo approaches lies in t he number of t ubes used when
performing t he procedure. The t wo-st ep react ion requires t hat t he reverse t ranscript ase
react ion and PCR amplificat ion be performed in separat e t ubes. The disadvant age of t he
t wo-st ep approach is suscept ibilit y t o cont aminat ion due t o more frequent sample
handling.[19] On t he ot her hand, t he ent ire react ion from cDNA synt hesis t o PCR amplificat ion
occurs in a single t ube in t he one-st ep approach. The one-st ep approach is t hought t o
minimize experiment al variat ion by cont aining all of t he enzymat ic react ions in a single
environment . It eliminat es t he st eps of pipet t ing cDNA product , which is labor-int ensive and
prone t o cont aminat ion, t o PCR react ion. The furt her use of inhibit or-t olerant polymerases,
polymerase enhancers wit h an opt imized one-st ep RT-PCR condit ion, support s t he reverse
t ranscript ion of t he RNA from unpurified or crude samples, such as whole blood and
serum.[20][21] However, t he st art ing RNA t emplat es are prone t o degradat ion in t he one-st ep
approach, and t he use of t his approach is not recommended when repeat ed assays from t he
same sample is required. Addit ionally, t he one-st ep approach is report ed t o be less accurat e
compared t o t he t wo-st ep approach. It is also t he preferred met hod of analysis when using
DNA binding dyes such as SYBR Green since t he eliminat ion of primer-dimers can be achieved
t hrough a simple change in t he melt ing t emperat ure. Nevert heless, t he one-st ep approach is
a relat ively convenient solut ion for t he rapid det ect ion of t arget RNA direct ly in biosensing.
End-point RT-PCR vs real-time RT-PCR

Quant ificat ion of RT-PCR product s can largely be divided int o t wo cat egories: end-point and
real-t ime.[22] The use of end-point RT-PCR is preferred for measuring gene expression
changes in small number of samples, but t he real-t ime RT-PCR has become t he gold st andard
met hod for validat ing quant it at ive result s obt ained from array analyses or gene expression
changes on a global scale.[23]

End-point RT-PCR

The measurement approaches of end-point RT-PCR requires t he det ect ion of gene
expression levels by t he use of fluorescent dyes like et hidium bromide,[24][25] P32 labeling of
PCR product s using phosphorimager,[26] or by scint illat ion count ing.[18] End-point RT-PCR is
commonly achieved using t hree different met hods: relat ive, compet it ive and
comparat ive.[27][28]

Relative RT-PCR
Relat ive quant ificat ions of RT-PCR involves t he co-amplificat ion of an int ernal cont rol
simult aneously wit h t he gene of int erest . The int ernal cont rol is used t o normalize t he
samples. Once normalized, a direct comparison of relat ive t ranscript abundances across
mult iple samples of mRNA can be made. One precaut ion t o not e is t hat t he int ernal cont rol
must be chosen so t hat it is not affect ed by t he experiment al t reat ment . The expression
level should be const ant across all samples and wit h t he mRNA of int erest for t he result s
t o be accurat e and meaningful. Because t he quant ificat ion of t he result s are analyzed by
comparing t he linear range of t he t arget and cont rol amplificat ion, it is crucial t o t ake int o
considerat ion t he st art ing t arget molecules concent rat ion and t heir amplificat ion rat e prior
t o st art ing t he analysis. The result s of t he analysis are expressed as t he rat ios of gene
signal t o int ernal cont rol signal, which t he values can t hen be used for t he comparison
bet ween t he samples in t he est imat ion of relat ive t arget RNA expression.[25][28][29]
Competitive RT-PCR
Compet it ive RT-PCR t echnique is used for absolut e quant ificat ion. It involves t he use of a
synt het ic “compet it or” RNA t hat can be dist inguished from t he t arget RNA by a small
difference in size or sequence. It is import ant for t he design of t he synt het ic RNA be
ident ical in sequence but slight ly short er t han t he t arget RNA for accurat e result s. Once
designed and synt hesized, a known amount of t he compet it or RNA is added t o
experiment al samples and is co-amplified wit h t he t arget using RT-PCR. Then, a
concent rat ion curve of t he compet it or RNA is produced and it is used t o compare t he RT-
PCR signals produced from t he endogenous t ranscript s t o det ermine t he amount of t arget
present in t he sample.[28][30]
Comparative RT-PCR
 Comparat ive RT-PCR is similar t o t he compet it ive RT-PCR in t hat t he t arget RNA
compet es for amplificat ion reagent s wit hin a single react ion wit h an int ernal st andard of
unrelat ed sequence. Once t he react ion is complet e, t he result s are compared t o an
ext ernal st andard curve t o det ermine t he t arget RNA concent rat ion. In comparison t o t he
relat ive and compet it ive quant ificat ion met hods, comparat ive RT-PCR is considered t o be
t he more convenient met hod t o use since it does not require t he invest igat or t o perform a
pilot experiment ; in relat ive RT-PCR, t he exponent ial amplificat ion range of t he mRNA must
be predet ermined and in compet it ive RT-PCR, a synt het ic compet it or RNA must be
synt hesized.[28][31][32][33][34]

Real-time RT-PCR

The emergence of novel fluorescent DNA labeling t echniques in t he past few years has
enabled t he analysis and det ect ion of PCR product s in real-t ime and has consequent ly led t o
t he widespread adopt ion of real-t ime RT-PCR for t he analysis of gene expression. Not only is
real-t ime RT-PCR now t he met hod of choice for quant ificat ion of gene expression, it is also
t he preferred met hod of obt aining result s from array analyses and gene expressions on a
global scale. Current ly, t here are four different fluorescent DNA probes available for t he real-
t ime RT-PCR det ect ion of PCR product s: SYBR Green, TaqMan, molecular beacons, and
scorpion probes. All of t hese probes allow t he det ect ion of PCR product s by generat ing a
fluorescent signal. While t he SYBR Green dye emit s it s fluorescent signal simply by binding t o
t he double-st randed DNA in solut ion, t he TaqMan probes', molecular beacons' and scorpions'
generat ion of fluorescence depend on Först er Resonance Energy Transfer (FRET) coupling of
t he dye molecule and a quencher moiet y t o t he oligonucleot ide subst rat es.[35]

SYBR Green
When t he SYBR Green binds t o t he double-st randed DNA of t he PCR product s, it will emit
light upon excit at ion. The int ensit y of t he fluorescence increases as t he PCR product s
accumulat e. This t echnique is easy t o use since designing of probes is not necessary given
lack of specificit y of it s binding. However, since t he dye does not discriminat e t he double-
st randed DNA from t he PCR product s and t hose from t he primer-dimers, overest imat ion of
t he t arget concent rat ion is a common problem. Where accurat e quant ificat ion is an
absolut e necessit y, furt her assay for t he validat ion of result s must be performed.
Nevert heless, among t he real-t ime RT-PCR product det ect ion met hods, SYBR Green is t he
most economical and easiest t o use.[22][23]
Taqman probes

TaqMan probes
TaqMan probes are oligonucleot ides t hat have a fluorescent probe at t ached t o t he 5' end
and a quencher t o t he 3' end. During PCR amplificat ion, t hese probes will hybridize t o t he
t arget sequences locat ed in t he amplicon and as polymerase replicat es t he t emplat e wit h
TaqMan bound, it also cleaves t he fluorescent probe due t o polymerase 5'- nuclease
act ivit y. Because t he close proximit y bet ween t he quench molecule and t he fluorescent
probe normally prevent s fluorescence from being det ect ed t hrough FRET, t he decoupling
result s in t he increase of int ensit y of fluorescence proport ional t o t he number of t he probe
cleavage cycles. Alt hough well-designed TaqMan probes produce accurat e real-t ime RT-
PCR result s, it is expensive and t ime-consuming t o synt hesize when separat e probes must
be made for each mRNA t arget analyzed.[22][16][36] Addit ionally, t hese probes are light
sensit ive and must be carefully frozen as aliquot s t o prevent degradat ion.
Molecular beacon probes
Similar t o t he TaqMan probes, molecular beacons also make use of FRET det ect ion wit h
fluorescent probes at t ached t o t he 5' end and a quencher at t ached t o t he 3' end of an
oligonucleot ide subst rat e. However, whereas t he TaqMan fluorescent probes are cleaved
during amplificat ion, molecular beacon probes remain int act and rebind t o a new t arget
during each react ion cycle. When free in solut ion, t he close proximit y of t he fluorescent
probe and t he quencher molecule prevent s fluorescence t hrough FRET. However, when
molecular beacon probes hybridize t o a t arget , t he fluorescent dye and t he quencher are
separat ed result ing in t he emit t ance of light upon excit at ion. As is wit h t he TaqMan probes,
molecular beacons are expensive t o synt hesize and require separat e probes for each RNA
t arget .[19]
Scorpion probes
 The scorpion probes, like molecular beacons, will not be fluorescent act ive in an
unhybridized st at e, again, due t o t he fluorescent probe on t he 5' end being quenched by t he
moiet y on t he 3' end of an oligonucleot ide. Wit h Scorpions, however, t he 3' end also
cont ains sequence t hat is complement ary t o t he ext ension product of t he primer on t he 5'
end. When t he Scorpion ext ension binds t o it s complement on t he amplicon, t he Scorpion
st ruct ure opens, prevent s FRET, and enables t he fluorescent signal t o be measured.[37]
Multiplex probes
TaqMan probes, molecular beacons, and scorpions allow t he concurrent measurement of
PCR product s in a single t ube. This is possible because each of t he different fluorescent
dyes can be associat ed wit h a specific emission spect ra. Not only does t he use of
mult iplex probes save t ime and effort wit hout compromising t est ut ilit y, it s applicat ion in
wide areas of research such as gene delet ion analysis, mut at ion and polymorphism analysis,
quant it at ive analysis, and RNA det ect ion, make it an invaluable t echnique for laborat ories of
many discipline.[37][38][39]

Two st rat egies are commonly employed t o quant ify t he result s obt ained by real-t ime RT-
PCR; t he st andard curve met hod and t he comparat ive t hreshold met hod.[40]

Application

The exponent ial amplificat ion via reverse t ranscript ion polymerase chain react ion provides for
a highly sensit ive t echnique in which a very low copy number of RNA molecules can be
det ect ed. RT-PCR is widely used in t he diagnosis of genet ic diseases and, semiquant it at ively,
in t he det erminat ion of t he abundance of specific different RNA molecules wit hin a cell or
t issue as a measure of gene expression.

Research methods

RT-PCR is commonly used in research met hods t o measure gene expression. For example, Lin
et al. used qRT-PCR t o measure expression of Gal genes in yeast cells. First , Lin et al.
engineered a mut at ion of a prot ein suspect ed t o part icipat e in t he regulat ion of Gal genes.
This mut at ion was hypot hesized t o select ively abolish Gal expression. To confirm t his, gene
expression levels of yeast cells cont aining t his mut at ion were analyzed using qRT-PCR. The
researchers were able t o conclusively det ermine t hat t he mut at ion of t his regulat ory prot ein
reduced Gal expression.[41] Nort hern blot analysis is used t o st udy t he RNA's gene expression
furt her.

Gene insertion
RT-PCR can also be very useful in t he insert ion of eukaryot ic genes int o prokaryot es.
Because most eukaryot ic genes cont ain int rons, which are present in t he genome but not in
t he mat ure mRNA, t he cDNA generat ed from a RT-PCR react ion is t he exact (wit hout regard
t o t he error-prone nat ure of reverse t ranscript ases) DNA sequence t hat would be direct ly
t ranslat ed int o prot ein aft er t ranscript ion. When t hese genes are expressed in prokaryot ic
cells for t he sake of prot ein product ion or purificat ion, t he RNA produced direct ly from
t ranscript ion need not undergo splicing as t he t ranscript cont ains only exons. (Prokaryot es,
such as E. coli, lack t he mRNA splicing mechanism of eukaryot es).

Genetic disease diagnosis

RT-PCR can be used t o diagnose genet ic disease such as Lesch–Nyhan syndrome. This
genet ic disease is caused by a malfunct ion in t he HPRT1 gene, which clinically leads t o t he
fat al uric acid urinary st one and sympt oms similar t o gout .[6] Analyzing a pregnant mot her and
a fet us for mRNA expression levels of HPRT1 will reveal if t he mot her is a carrier and if t he
fet us will likely t o develop Lesch–Nyhan syndrome.[42]

Cancer detection

Scient ist s are working on ways t o use RT-PCR in cancer det ect ion t o help improve prognosis,
and monit or response t o t herapy. Circulat ing t umor cells produce unique mRNA t ranscript s
depending on t he t ype of cancer. The goal is t o det ermine which mRNA t ranscript s serve as
t he best biomarkers for a part icular cancer cell t ype and t hen analyze it s expression levels
wit h RT-PCR.[43]

RT-PCR is commonly used in st udying t he genomes of viruses whose genomes are composed
of RNA, such as Influenzavirus A, ret roviruses like HIV and SARS-CoV-2.[44]

Challenges

Despit e it s major advant ages, RT-PCR is not wit hout drawbacks. The exponent ial growt h of
t he reverse t ranscribed complement ary DNA (cDNA) during t he mult iple cycles of PCR
produces inaccurat e end point quant ificat ion due t o t he difficult y in maint aining linearit y.[45] In
order t o provide accurat e det ect ion and quant ificat ion of RNA cont ent in a sample, qRT-PCR
was developed using fluorescence-based modificat ion t o monit or t he amplificat ion product s
during each cycle of PCR. The ext reme sensit ivit y of t he t echnique can be a double edged
sword since even t he slight est DNA cont aminat ion can lead t o undesirable result s.[46] A
simple met hod for eliminat ion of false posit ive result s is t o include anchors, or t ags, t o t he 5'
region of a gene specific primer.[47] Addit ionally, planning and design of quant ificat ion st udies
can be t echnically challenging due t o t he exist ence of numerous sources of variat ion
including t emplat e concent rat ion and amplificat ion efficiency.[31] Spiking in a known quant it y
of RNA int o a sample, adding a series of RNA dilut ions generat ing a st andard curve, and adding
in a no t emplat e copy sample (no cDNA) may used as cont rols.

Protocol

RT-PCR can be carried out by t he one-st ep RT-PCR prot ocol or t he t wo-st ep RT-PCR
prot ocol.

One-step RT-PCR

One-st ep RT-PCR subject s mRNA t arget s (up t o 6 kb) t o reverse t ranscript ion followed by
PCR amplificat ion in a single t est t ube. It is import ant t o not e t hat using int act , high qualit y
RNA and a sequence-specific primer will produce t he best result s.

Once a one-st ep RT-PCR kit wit h a mix of reverse t ranscript ase, Taq DNA polymerase, and a
proofreading polymerase is select ed and all necessary mat erials and equipment are obt ained
a react ion mix is t o be prepared. The react ion mix includes dNTPs, primers, t emplat e RNA,
necessary enzymes, and a buffer solut ion. The react ion mix is added t o a PCR t ube for each
react ion, followed by t emplat e RNA. The PCR t ubes are t hen placed in a t hermal cycler t o
begin cycling. In t he first cycle, synt hesis of cDNA occurs. The second cycle is t he init ial
denat urat ion wherein reverse t ranscript ase is inact ivat ed. The remaining 40-50 cycles are t he
amplificat ion, which includes denat urat ion, annealing, and elongat ion. When amplificat ion is
complet e, t he RT-PCR product s can be analyzed wit h gel elect rophoresis.[48][49]

(PCR Applicat ions Manual and Biot ools)

Two-step RT-PCR

Two-st ep RT-PCR, as t he name implies, occurs in t wo st eps. First t he reverse t ranscript ion
and t hen t he PCR. This met hod is more sensit ive t han t he one-st ep met hod. Kit s are also
useful for t wo-st ep RT-PCR. Just as for one-st ep PCR, use only int act , high qualit y RNA for
t he best result s. The primer for t wo-st ep PCR does not have t o be sequence specific.

Step one
First combine t emplat e RNA, primer, dNTP mix, and nuclease-free wat er in a PCR t ube. Then,
add an RNase inhibit or and reverse t ranscript ase t o t he PCR t ube. Next , place t he PCR t ube
int o a t hermal cycler for one cycle wherein

annealing, ext ending, and inact ivat ing of reverse t ranscript ase occurs. Finally, proceed direct ly
t o st ep t wo which is PCR or st ore product on ice unt il PCR can be performed.

Step two

Add mast er mix which cont ains buffer, dNTP mix, MgCl2, Taq polymerase and nuclease-free
wat er t o each PCR t ube. Then add t he necessary primer t o t he t ubes. Next , place t he PCR
t ubes in a t hermal cycler for 30 cycles of t he amplificat ion program. This includes:
denat urat ion, annealing, and elongat ion. The product s of RT-PCR can be analyzed wit h gel
elect rophoresis.[50]

Publication guidelines

Quant it at ive RT-PCR assay is considered t o be t he gold st andard for measuring t he number
of copies of specific cDNA t arget s in a sample but it is poorly st andardized.[51] As a result ,
while t here are numerous publicat ions ut ilizing t he t echnique, many provide inadequat e
experiment al det ail and use unsuit able dat a analysis t o draw inappropriat e conclusions. Due t o
t he inherent variabilit y in t he qualit y of any quant it at ive PCR dat a, not only do reviewers have
a difficult t ime evaluat ing t hese manuscript s, but t he st udies also become impossible t o
replicat e.[52] Recognizing t he need for t he st andardizat ion of t he report ing of experiment al
condit ions, t he Minimum Informat ion for Publicat ion of Quant it at ive Real-Time PCR
Experiment s (MIQE, pronounced mykee) guidelines have been published by an int ernat ional
consort ium of academic scient ist s. The MIQE guidelines describe t he minimum informat ion
necessary for evaluat ing quant it at ive PCR experiment s t hat should be required for
publicat ion for encouraging bet t er experiment al pract ice and ensuring t he relevance,
accuracy, correct int erpret at ion, and repeat abilit y of quant it at ive PCR dat a.[53]

Besides report ing guidelines, t he MIQE st resses t he need t o st andardize t he nomenclat ure
associat ed wit h quant it at ive PCR t o avoid confusion; for example, the abbreviation qPCR
should be used for quantitative real-time PCR and RT-qPCR should be used for reverse
transcription-qPCR, and genes used for normalisat ion should be referred t o as reference
genes inst ead of housekeeping genes. It also proposes t hat commercially derived t erms like
TaqMan probes should not be used but inst ead referred t o as hydrolysis probes. Addit ionally,
it is proposed t hat quant ificat ion cycle (Cq) be used t o describe t he PCR cycle used for
quant ificat ion inst ead of t hreshold cycle (Ct ), crossing point (Cp), and t akeoff point (TOP),
which refer t o t he same value but were coined by different manufact urers of real-t ime
inst rument s.[51]

The guideline consist s of t he following element s: 1) experiment al design, 2) sample, 3)

nucleic acid ext ract ion, 4) reverse t ranscript ion, 5) qPCR t arget informat ion, 6)
oligonucleot ides, 7) prot ocol, 8) validat ion, and 9) dat a analysis. Specific it ems wit hin each
element carry a label of eit her E (essent ial) or D (desirable). Those labelled E are considered
crit ical and indispensable while t hose labelled D are considered peripheral yet import ant for
best -pract ices.[53]

References

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External links

RT-PCR prot ocols from Penn st at e Universit y (ht t p://www.cas.psu.edu/docs/CASDEPT/VE

T/jackvh/jvhpcr.ht ml)

Dat abase of validat ed PCR primer set s (ht t p://www.realt imeprimers.org) (websit e
crit ique (ht t p://www.genengnews.com/best ofweb/list .aspx?iid=93) )

Animat ion t o illust rat e RT-PCR procedure, from Cold Spring Harbor Laborat ory (ht t p://www.
bio.davidson.edu/Courses/immunology/Flash/RT_ PCR.ht ml)

The Reference in qPCR – an Academic & Indust rial Informat ion Plat form (ht t p://www.gene-
quant ificat ion.info/)

Top 5 Government Rt Pcr Cent res in Mumbai (ht t ps://www.t eckoct ane.com/t op-5-govern
ment -rt -pcr-t est ing-cent res-in-mumbai-wit h-cont act -det ails-and-locat ion/)

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