WWHHYY RReeAALL--TTIIMMee PPCCRR??
DISADvANTAge Of TRADITIONAL PCR
Poor precision (Northern could even be
Results are not expressed as numbers
Ethidium bromide staining is not very
WWHHYY RReeAALL--TTIIMMee PPCCRR??
• ADvANTAgeS Of ReAL-TIMe PCR
amplification can be monitored real-time
wider dynamic range of up to 1010-fold
no post-PCR processing of products
(No gel-based analysis at the end of the PCR
reaction Þ high throughput)
ultra-rapid cycling (30 minutes to 2 hours)
WWHHAATT IISS RRTT--PPCCRR??
• Cells in all organisms regulate gene expression by turnover
of gene transcripts (messenger RNA, abbreviated to
mRNA): The amount of an expressed gene in a cell can be
measured by the number of copies of an mRNA transcript
of that gene present in a sample.
• RT PCR reaction is a laboratory technique of molecular
biology based on the polymerase chain reaction (PCR),
which is used to amplify and simultaneously detect or
quantify a targeted DNA molecule.
• During this reaction, fluoroprobes bind to specific target
regions of amplicons to produce fluorescence during PCR.
The fluorescence, measured in Real Time, is detected in a
PCR cycler with an inbuilt filter flurometer.
WHAT AARRee ffLLuuOORReeSSCCeeNNTT
• When a population of fluorochrome molecules is excited by
light of an appropriate wavelength, fluorescent light is
emitted. The light intensity can be measured by flurometer
or a pixel-by-pixel digital image of the sample.
• Excitation and Emission: Fluorodyes absorb light at one
wavelength & thereby boosts an electron to a higher
• The excited electron falls back to the ground state and the
flurophore re- emits light but at longer wavelength.
• This shift makes it possible to separate excitation light from
emission light with the use of optical filters.
• The wavelength (nm) where photon energy is most
efficiently captured is defined as the Absorbancemax & the
wavelength (nm) where light is most efficiently released is
defined as the Emissionmax.
WHAT IISS ffLLuuOORReeSSCCeeNNCCee
• FRET is a distance dependent interaction between the
excited states of 2 dye molecules in which excitation is
transferred from a donor molecule to an acceptor
molecule without emission of a photon.
• A donor chromophore, initially in its electronic excited
state, then transfer energy to an acceptor
chromophore. The efficiency of this energy transfer is
inversely proportional to the sixth power of the
distance (1 / r6) between donor and acceptor, making
FRET extremely sensitive to small changes in distance.
1. Denaturation step: This step consists of heating the
reaction to 90–95 °C. It causes DNA separation by
disrupting the hydrogen bonds between complementary
bases, yielding single strands of DNA.
2. Annealing step: The reaction temperature is lowered to
50–65 °C allowing hybridization of the primers to the
single-stranded DNA template.
3. Extension/Elongation step: At this step, the Taq
polymerase synthesizes a new DNA strand complementary
to the DNA template strand by adding dNTPs that are
complementary to the template in 5' to 3' direction.
PROTOCOLS IINN RReeAALL--TTIIMMee
• For analysis of gene expression levels, the RNA first needs
to be transcribed into cDNA using a reverse transcriptase.
Reverse transcriptases are enzymes generally derived
from RNA-containing retroviruses. RT-PCR can take place
in a two-step or one-step reaction.
• With two-step RT-PCR, the RNA is first reverse transcribed
into cDNA using oligo-dT primers, random oligomers, or
gene-specific primers. An aliquot of the reverse-transcription
reaction is then subsequently added to the
• Precious RNA samples can be immediately transcribed
into more stable cDNA for later use and long-term storage
potocolss iinn rreeaall--ttiimmee
• In one-step RT-PCR — also referred to as one-tube RT-PCR
— both reverse transcription and amplification take
place in the same tube, with reverse transcription
preceding PCR. This is possible due to specialized reaction
chemistries and cycling protocols. The fast procedure
enables rapid processing of multiple samples and is easy
• The reduced number of handling steps results in high
reproducibility from sample to sample and minimizes the
risk of contamination since less manipulation is required.
The advantages of each method are given below.
Two-step RT-PCR One-step RT-PCR
■ Multiple PCRs from one RT reaction ■ Easy handling
■ Flexibility with RT primer choice ■ Fast procedure
■ Enables long-term storage of cDNA ■ High reproducibility
■ Low contamination risk
• When the SYBR Green binds to the double-stranded DNA
of the PCR products, it will emit light upon excitation. The
intensity of the fluorescence increases as the PCR
• This technique is easy to use since designing of probes is
not necessary given lack of specificity of its binding.
• However, since the dye does not discriminate the double-stranded
DNA from the PCR products and those from the
primer-dimers, overestimation of the target concentration is
a common problem. Where accurate quantification is an
absolute necessity, further assay for the validation of
results must be performed.
• Nevertheless, amongst the real-time RT-PCR product
detection methods, SYBR Green is the most economical
and easiest to use.
• The two most popular alternatives to SYBR Green are
TaqMan and molecular beacons, both of which are relying
on FRET for quantitation.
• TaqMan probes are oligonucleotides that have a
fluorescent probe attached to the 5' end and a quencher to
the 3' end.
• During PCR amplification, these probes will hybridize to the
target sequences located in the amplicon and as
polymerase replicates the template with TaqMan bound, it
also cleaves the fluorescent probe due to polymerase 5'-
• This separates the fluorescent and quenching dyes and
FRET no longer occurs. Fluorescence increases in each
cycle, proportional to the rate of probe cleavage.
• Similar to the TaqMan probes, Molecular Beacons also
make use of fluorescent probes attached to the 5' end and
a quencher attached to the 3' end of an oligonucleotide
substrate, but FRET only occurs when the quenching dye
is directly adjacent to the fluorescent dye.
• However, whereas the TaqMan fluorescent probes are
cleaved during amplification, Molecular Beacon probes
remain intact and rebind to a new target during each
• When free in solution, the close proximity of the fluorescent
probe and the quencher molecule prevents fluorescence.
• When Molecular Beacon probes hybridize to a target, the
fluorescent dye and the quencher are separated resulting
in the emittance of light upon excitation.
• The Scorpion probes, like Molecular Beacon, will not be
fluorescent active in an unhybridized state, again, due to
the fluorescent probe on the 5' end being quenched by the
moiety on the 3' end of an oligonucleotide.
• With Scorpions, the probe and primer present as one
molecule. However, the 3' end contain a primer, the 5' end
contain a hairpin structure of probe and the loop is
complementary to the target.
• A PCR blockers lies between the primer and the probe
prevent the polymerase copying the probe.
• After amplification, heating cause the 3 primers to unfold,
then cooling allowing the complementary probe to aneal
which prevent the hairpin reformming again and then
separate the fluorophore and quencher releasing
• TaqMan probes, Molecular Beacons and Scorpions allow
the concurrent measurement of PCR products in a single
tube. This is possible because each of the different
fluorescent dyes can be associated with a specific
• The term multiplex refers to the fact that detection of more
than one pathogen is carried out simultaneously in one
tube, by simultaneously amplifying multiple sequences in
one single reaction.
• Not only does the use of multiplex probes save time and
effort without compromising test utility, its application in
wide areas of research such as gene deletion analysis,
mutation and polymorphism analysis, quantitative analysis,
and RNA detection, make it an invaluable technique for
laboratories of many discipline.
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• Baseline: The baseline is the noise level in early cycles,
typically measured between cycles 3 and 15, where there
is no detectable increase in fluorescence due to
• Background: This refers to nonspecific fluorescence in the
• example, due to inefficient quenching of the fluorophore or
the presence of large amounts of double-stranded DNA
template when using SYBR Green I. The background
component of the signal is mathematically removed by the
software algorithm of the real-time cycler.
• Reporter signal: Fluorescent signal that is generated during
real-time PCR by either SYBR Green I or by a fluorescently
labeled sequencespecific probe.
baSic tteerrMMSS uuSSeedd iinn rrtt--
• Normalized reporter signal (Rn): This is the emission
intensity of the reporter dye divided by the emission
intensity of the passive reference dye measured in each
• Passive reference dye: On some real-time PCR
instruments, the fluorescent dye ROX serves as an internal
reference for normalization of the fluorescent signal.
• Threshold: The threshold is adjusted to a value above the
background and significantly below the plateau of an
• Threshold cycle (CT) or crossing point: This is the cycle at
which the amplification plot crosses the threshold, i.e., at
which there is a significant detectable increase in
fluorescence. The CT serves as a tool for calculation of the
starting template amount in each sample
baSic tteerrMMSS uuSSeedd iinn rrtt--
• Endogenous reference gene: This is a gene whose
expression level should not differ between samples, such
as a housekeeping or maintenance gene.
• The use of an endogenous reference gene corrects for
variation in RNA content, variation in reverse-transcription
efficiency, possible RNA degradation or presence of
inhibitors in the RNA sample, variation in nucleic acid
recovery, and differences in sample handling.
• Calibrator sample: This is a reference sample used in
• Standard curve: it is commonly generated using a dilution
series of at least 5 different concentrations of the standard.
Real Time PPCCRR iinnssTTRRuummeennTTss
1a. Excitation filters
1b. Emission filters
2. Tungsten halogen light source
(350 - 1000nm continuous)
Real Time Detection
3. Microplate format
33.. TTyyPPeess ooff RReeaall--TTiimmee PPCCRR
* Absolute quantification
It uses serially diluted standards of known concentrations to
generate a standard curve. The standard curve produces a linear
relationship between Ct and initial amounts of total RNA or cDNA,
allowing the determination of the concentration of unknowns
based on their Ct values. This method assumes all standards and
samples have approximately equal amplification efficiencies
* Relative quantification (relative fold change)
Based on either an external standard or a reference sample, also known as a
calibrator. When using a calibrator, the results are expressed as a
i. Relative standard method (using a standard curve and
then expressed relative to a single calibrator sample)
ii. Comparative CT (2 -DDCT) method
3. PCR Quantification