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Lecture 2

The document discusses key considerations for real-time RT-PCR, including primer design, amplification efficiency, and reference gene selection. It emphasizes the importance of using target lengths of 100-200 bp for optimal efficiency and outlines methods for quantifying mRNA levels, such as the delta Ct and Pfaffl methods. Additionally, it provides calculations for efficiency and fold induction based on various approaches to data analysis.

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nicolas.troncoso.c
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2 views

Lecture 2

The document discusses key considerations for real-time RT-PCR, including primer design, amplification efficiency, and reference gene selection. It emphasizes the importance of using target lengths of 100-200 bp for optimal efficiency and outlines methods for quantifying mRNA levels, such as the delta Ct and Pfaffl methods. Additionally, it provides calculations for efficiency and fold induction based on various approaches to data analysis.

Uploaded by

nicolas.troncoso.c
Copyright
© © All Rights Reserved
Available Formats
Download as PDF, TXT or read online on Scribd
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CONSIDERATIONS IN REAL TIME RT-

PCR

Primer Design
Amplification Efficiency
For the highest efficiency in real-time RT-PCR using SYBR
Green, targets should ideally be 100–200 bp in length.

Reference Gene Selection


Importance of
Primers in PCR
specific
high efficiency
no primer-dimers
Ideally should not give a DNA signal
cross exon/exon boundary
CONSIDERATIONS IN REAL TIME RT-
PCR

Primer Design
Amplification Efficiency
For the highest efficiency in real-time RT-PCR using SYBR
Green, targets should ideally be 100–200 bp in length.

Reference Gene Selection


10,000,000,000
1,000,000,000 100% EFF
100,000,000 90% EFF
80% EFF
AMOUNT OF DNA

10,000,000
70% EFF
1,000,000
100,000
10,000
1,000
100
10
1
0 10 20 30
PCR CYCLE NUMBER
Efficency Calculation

Eff can be calculated by the formula: y = -3.465x + 34.005


35 R2 = 0.9968
Eff = 10(-1/slope) – 1 30

25

20
The efficiency of the PCR should be 15

90 - 100% (– 3.6 > slope > – - 10

5
3.1) 0

10^-(1/-3.4)= 1.96
0 1 2 3 4 5 6

(1.96-1)*100= 96% efficient


CONSIDERATIONS IN REAL TIME RT-
PCR

Primer Design
Amplification Efficiency
For the highest efficiency in real-time RT-PCR using SYBR
Green, targets should ideally be 100–200 bp in length.

Reference Gene Selection


CONSIDERATIONS IN REAL TIME RT-
PCR
Choosing a proper internal control (housekeeping) gene for
normalization.
The internal control gene(s) should not vary in the tissues or
cells under investigation.
Minimal number of most stable genes should be used.
For the averaging of the selected genes geometric mean is
more accurate than arithmetic.
Efficency Calculation

Eff can be calculated by the formula:


Eff = 10-1/slope – 1

y = -3.465x + 34.005
35 R2 = 0.9968
The efficiency of the PCR should be 30

90 - 100% (– 3.6 > slope > – - 25

20
3.1) 15

10

10^-(1/-3.4)= 1.96
0
0 1 2 3 4 5 6

(1.96-1)*100= 96% efficient


QUANTITATION OF mRNA LEVELS USING
REAL TIME PCR

STANDARD CURVE METHOD (absolute)


Basic delta Ct method
Delta-Delta CT METHOD (An approximation method
relative)
2-(Ct)
Ct:[(Cttumor-Cthousekeeping)-(Ctnormal-Cthousekeeping)]

PFAFFL METHOD
STANDARD CURVE
METHOD
‘copy number’ target gene control

Dilution curve target gene

‘copy number’ target gene experimental

fold change in target gene=


copy number experimental
copy number control
Calculating for relative quantitation

Basic delta Ct method:

(no normalization to reference gene)


Primer set #2
Tissue #1: 22
Tissue (control) #2: 24

Delta Ct: 24-22 = 2

Fold induction = 22 = 4
Calculating for relative quantitation

Delta-delta Ct method:

(assumes same efficiencies for each primer set)

Reference Primer set HouseKe. Primer set


Tissue #1: 21 22
Tissue (control) #2: 20 24

Delta Ct: 22-21 = 1


1st Delta
Delta Ct: 24-20 = 4

2nd Delta Delta Ct: 4-1 = 3

Fold induction = 23 = 8
Calculating for relative quantitation

Pfaffl method:

(Pfaffl, 2001; Nucleic Acid Research)

deltaCt target (control-sample)


Efficiencytarget
Fold induction =
deltaCt reference (control-sample)
Efficiencyreference

Efficiency = 10-1/slope
Calculating for relative quantitation

Pfaffl method:
(efficiencies are normalized)
Primer set #1Reference Primer set #2 HouseKe
Tissue #1: 21 22

Tissue (control) #2: 20 24

(From Standard curve) Efficiency: 90% = 1.9 100% = 2


Delta Ct: 20-21 = -1 24-22 = 2

deltaCt target (24-22 = 2)


2target 4
Fold induction = = = 7.5
deltaCt reference (20-21 = -1)
1.9reference 0.53
Comparison of methods for relative
quantitation calculations
Basic delta Ct method: (no reference gene)
Fold induction : 4

Delta-delta Ct method: (reference gene)


Fold induction : 8
Ideal for primer pairs with an E ≥ 90% AND large fold changes in
expression (10 fold or more)

Pfaffl method: (reference gene and efficiency)


Fold induction : 7.5

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