qRT-PCR.pdf

Reverse Transcription Real- time quantitative PCR

1. Reverse Transcription Real-
time quantitative PCR
(qRT-PCR)

2. Central Dogma of Molecular Biology
TranscriptionTranslation
Technologies
used

3. Transcription Analysi
•Transcription:istheprocessbywhichtheinformationinastrandofDNAis
copiedintoanewmoleculeofmessengerRNA(mRNA),thismRNAisneeded
tocarrytheinformationneededtobuildapolypeptideaswellascontrolling
whichproteinneedtobeproducedineachcellandinwhichquantity.
•Why studying RNA is important?
-To find which genes are active (expressed) in a particular cell type and the
level of the genes activity.
•Technology used in measuring transcription:
1-Northern
2-qRT-PCR
4-Microarray
5-RNA-Seq

4. 1-Background
on Basic PCR

5. Cofactor of polymerase
Polymerase
MgCl
dNTPs
Primer
Reagent
Background infromation of PCR

6. Amplicons (product) Detection after PCR
●Detection is done by runing Gel Electrophoresis only after the PCR run is finished.

7. 1-Introduction
of qRT-PCR

8. What is qRT-PCR ?
●ReverseTranscriptionReal-time
quantitativePCR(qRT-PCR)isthemost
commonlyusedtechnologyforstudying
transcriptionprocesses.
●Itisbasedonreliabledetectionand
measurementofproductsgenerated
duringeachcycleofthePCRprocess,
whicharedirectlyproportionaltothe
amountoftemplatepriortothestartof
thePCRprocess.
Terms:
q=quantitativeàitgives
quantitativemeasurementof
theRNAsample,thusgivesan
ideaaboutthelevelof
expression.
RT=Realtimeàdetectingthe
productinarealtimeduring
eachcycleofthePCR.
RT=ReverseTranscriptionà
byusingReversetranscriptase
enzymethatconvertRNAinto
complementaryDNA(cDNA).

9. qRT-PCR
Polymerase
MgCl
dNTPs
Primers
Reagent
Fluorescent dye
Reporter
Reverse
transcriptase

10. Majordiffrence between PCR and qRT-PCR
●PCR ●qRT-PCR
●Detection of amplicons is done after the
PCR runby runing Gel electrophoresis
●Detection of amplicons presense and
concentration is done during the run in Real
Time (RT)by sectrflurometry.
●All techniques can generates copies of a DNA or RNA template exponentially using thermalcycler.

11. 3-Steps of
qRT-PCR

12. 3-Extension
Steps of Reverse Transcription Real Time PCR
•RNA is converted into complementary DNA (cDNA)
throughReverse transcriptaseenzyme.
•Primer complementary to RNA molecule is
annealed at low temperature then a higher
temperature is used to activate Reverse
transcriptaseenzyme.
•Why we need to convert single strand RNA into the
double strand cDNA?
ssRNAis fragile molecules while cDNA is more stable.
A. Reverse Transcription

13. 3-Extension
1-Denaturation
2-Annealing
Steps of Reverse Transcription Real Time PCR
High temperature incubation is used to
“melt” double-stranded cDNAinto single
strands.
During annealing, specific primers of
targeted region have an opportunity to
hybridize.
At 70-72°C, the activity of the DNA
polymerase is optimal, and primer
extension occurs at rates of up to 100
bases per second.
B. Amplification

14. •The detection is based on fluorescence
technology.
•The instruments in the Real Time PCR is
subjected to light(tungsten or halogen)
source
•the reporteradded to the sample
fluoresce due to the excitation by the light
source.
•the signal is amplified with the
amplification of copy number of sample
cDNA.
•The emitted signal is detected by a
detector.
Steps of Real Time PCR
C. Detection

15. 4-Types of
qRT-PCR

16. Fluorescent Dye-
Based RT-PCR
SYBR Green
Scorpions
Dual
hybridization
probes
Molecular
beacons
Fluorescent Probe-
Based RT-PCR
Taq Man
The various available chemistries for real-time PCR

17. Real-Time
quantitative
TaqMan® assay
FluorescentProbe based
qRT-PCR

18. TaqMan qRT-PCR
Polymerase
MgCl
dNTPs
Primers
Reagent
Reporter
oligonucleotide probe
Forward and Reverse

19. Fluorescent oligonucleotide Probe
●Anoligonucleotideprobe,whichwasdesigned
tohybridizewithinthetargetsequenceis
introducedintothePCRassay.
●Theprobehasareporterfluorescentdyeatthe
5 ́endandaquencherattachedtothe3 ́end.
Oligonucleotide probe complementary
to a specific sequence within the
target region.
5’3’
Fluorescent ReporterQuencher

20. Fluorescent oligonucleotide Probe
●Whenprobeisintactàthecloseproximityofthe
quenchertothereportersignificantlydecreasesthe
fluorescenceemittedbythereporterdye.
àNofluorescentisemitted
●WhenprobeiscleavedàAfluorescencesignalis
emitted.
àfluorescentisemitted

21. Principle of TaqMan qRT-PCR

22. How does the Taq polymerase cleave my probe?
●BecauseThermusaquaticus(i.e.,Taq)polymerasehas5 ́to3 ́exonuclease
activity.
●Whatdoes5 ́to3 ́exonucleaseactivitymean?
Istheprocessofremovingnucleotidesatthe5’
endofamolecule.
Taq Polymerase
CleavageofatargetprobeduringPCRbythe5 ́exnuclease
activityofTaqpolymerasecanbeusedtodetectamplificationof
thetarget-specificproduct.

23. Principle of TaqMan qRT-PCR
•Cleavage of the probe separates the
reporter and quencher dyes, thereby
emitting a fluorescence signal.
•Cleavage also removes the probe from the
target strand, allowing primer extension to
continue to the end of template strand,
thereby not interfering with the exponential
accumulation of PCR product.
•Ifthetargetsequenceispresent,the
fluorogenicprobeannealsdownstream
fromoneoftheprimersitesandis
cleavedbythe5 ́nucleaseactivityofthe
Taqpolymeraseenzymeduringthe
extensionphaseofthePCR.

24. TaqMan qRT-PCR Result
●Additional reporter dye molecules
are cleaved from their respective
probes with each cycle
●This leads to an increase in
fluorescence intensity
proportional to the amount of
amplicon produced.

25. SYBRgreen
qRT-PCR
Fluorescent Dye based
qRT-PCR

26. SYBR Green qRT-PCR
Polymerase
MgCl
dNTPs
Primers
Reagent
Reporter
Forward and Reverse
SYBRgreen dye

27. SYBR Green qRT-PCR
●In this method amplicon-specific labeled
oligonucleotide probeare not required.
●SYBR Green is a dye that emits prominent fluorescent
signal when it binds at the minor groove of the dsDNA
(nonspecifically).
●It intercalates into dsDNAonly (it does not bind to
single-stranded DNA).
Binding of SYBR Green
dye to the minor groove
of dsDNA

28. Principle of SYBR green qRT-PCR

29. Tag Man
SYBR Green
1
2
Comparisons between Tag Man andSYBR Green
Fluorescent Probe-Based RT-PCR
Fluorescent Dye-Based RT-PCR
Non spcific
Spcific
The usage of sequence specific oligonucleotide
fluorogenic probes leads to the elimination of
nonspecific result.
Cheap
Easyto produce
Sensitivesensitive to the minor groves of dsDNA
Simple
dye binds to all dsDNAsformed during the PCR
reaction (i.e., nonspecific PCR products and
primer-dimers).
Expensive

30. Multiplexing
in qRT-PCR

31. What is Multiplexing?
Multiplex PCR representsa variant of PCR in which two or more
DNA fragments are simultaneously amplified within a single
reaction tube. This is achieved by includingmore than one primer pair
to the reaction mixture

32. Qualtiy
Assurance in
qRT-PCR

33. Qualtiy Assurance
●Controls used:
●Internal control:
○The internal controls used in the qRT-PCR arehousekeeping genes, whose expression remains same
throughout the developmental stages, different tissues and differentenvironmental (experimental)
conditions.
○It is added to each sample to ensure that the PCR goes well and no random error has been made to
the sample.
○Random error like pipetting error or forgetting to add a reagent of the PCR reaction.
○It has to be always positive (present) in each unknown sample.
●Positive Control:a sperate tube where a known positive sample (of my RNA region) is added to the reagent.
○Has to give positiveresult always.
○In each run of qRT-PCR one positive control at least has to be included.
●Negative sample:a sperate tube where a known negative sample (of my RNA region) is added to the reagent.
○Has to give negativeresult always.
○In each run of qRT-PCR one negative control has to be included.

34. 5-Amplification
curve Analysis

35. 1-The linear ground phase:
●Startsusually at the first 10–15 cycles
●PCR is just beginning, and fluorescence
emission at each cycle has not yet risen
above background.
●Baselinefluorescence is calculated at this
time.
2-Early exponential phase:
●At this phase the amount of fluorescence
has reached a threshold where it is
significantly higher (usually 10 times the
standard deviation of the baseline) than
background levels.
●The cycle at which this occurs is known as
Ct.
●This value is representative of the starting
copy number in the original template and
is used to calculate experimental results.
Phases of the PCR amplification curve

36. 3-Loglinear (also known as exponential) phase:
●During this phase, PCR reaches its optimal
amplification period with the PCR product
doubling after every cycle in ideal reaction
conditions.
4-Plateau phase:
●The last phase is the plateau stage and is
reached when the reaction components
become limitedand the fluorescence
intensity is no longer useful for data
calculation.
Phases of the PCR amplification curve

37. Nomenclature of qRT-PCR Amplification Curve
∆Rn:
•The computer software program calculates a ∆Rn
using the equation
Rn = Rnf–Rnb
oRnf: is the fluorescence emission of the product at
each time point
oRnb: is the fluorescence emission of the baseline
•The ∆Rn values are plotted versus the cycle
number.
•During the early cycles of PCR amplification, ∆Rn
values do not exceed the baseline.

38. Threshold:
●The threshold is chosen by the software
or be manually adjusted, based on the
variability of the baseline.
●It is calculated as ten-times the standard
deviation of the average signal of the
baseline fluorescent signal between
cycles 3 to 15.
●A fluorescent signal that is detected
above the threshold is considered a real
signal that can be used to define the
thresholdcycle(Ct) for a sample.
Nomenclature of qRT-PCR Amplification Curve

39. Ct: ThresholdCycle
●Ct is the fractional PCR cycle number at
which the reporter fluorescence is greater
than the minimal detection level (i.e., the
threshold).
●The Ct is a basic principle of real-time PCR
and is an essential component in producing
accurate and reproducible data.
Nomenclature of qRT-PCR Amplification Curve

40. Positive VS Negative Result

41. Low Ct VS High Ct
Cycle Number
Thershold
Fluorescennce
0 15 25 35
Ct=25
àMore template present at the start of the
reaction
àfewer number of cycles needed to reach
the point at which the fluorescent signal is
recorded as statistically significant above
background or the baseline
àLow Ct value
Cycle Number
Thershold
Fluorescennce
0 15 25 35
Ct=39
àLess template present at the start of the
reaction
àHigher number of cycles needed to reach
the point at which the fluorescent signal is
recorded as statistically significant above
background or the baseline
àHigh Ct value

42. Thank you
Any Quations?