Download Real-time PCR

Real-Time PCR
(Quantitative PCR)
Goals
1. Understand the fundamental difference
between qPCR and traditional PCR
2. Understand the basic quantification method
using of qPCR
3. Understand differences between qPCR and
Northern blotting
Applications of real-time PCR
• Powerful and reliable quantitative method
– Gene expression
– Determination/monitoring of viral load
– Quantification of cancer genes
– Microarray verification
– Transgenic copy
– SNP analysis
Steps of real-time PCR
Amount of PCR product
• Three phases
Plateau phase
Linear phase
Exponential phase
PCR cycle number
Exponential amplification of PCR
Xn = X0 * (1 + E)
E=
(–1/slope)
[10
]
n
Xn = DNA copies at cycle n
X0 = DNA copies at cycle 0
E = efficiency of amplification
n = cycle number
–1
(Efficiency = 1 during exponential amplification)
Quantitative detection system
• Fluorescence detection system
• Two types of fluorochromes
– DNA binding dye
– Probe-based fluorochromes
SYBR green (DNA binding dye)
Most commonly
used
SYBR green
Probe-based fluorochromes
(FAM, VIC, TET, FRET)
Less commonly
used now
Fluorophore
Quencher
SYBR green
Vs.
• Does not discriminate
between the gene of
interest and other DNAs
(i.e. contamination)
• Does not allow to do
multiplex PCR
• Requires less steps
• Less costly
Probe-based Fluoro.
• Does discriminate,
more specific
• Allows multiplex PCR
with usage of different
fluoro.
• Requires multiple steps
• More costly
Detection zones qPCR vs PCR
Amount of PCR product
Traditional PCR with
EtBr
qPCR
PCR cycle number
Amplicon quantification by
qPCR
• Fluorescence increase is proportional to DNA
amplification
• The first cycle at which the instrument can
distinguish the amplified fluorescence as
being above the background level is called the
threshold cycle or “Ct”
The threshold cycle (Ct)
Example of a Ct curve
Ct
The threshold cycle (Ct)
Ct curves of three different samples.
The threshold cycle (Ct)
• The Ct value is inversely proportional to the
starting concentration of the sample
– i.e. the greater the amount of DNA in the sample
the lower the Ct value
Quantification methods
1. Absolute quantification
– To determine exact amounts of DNA (e.g. viral load)
2. Relative quantification
– To determine changes in gene expression
Absolute quantification
• If initial amount of DNA copies is known:
XT = X0 * (1 + E) Ct
XT = DNA copies at threshold
X0 = DNA copies at cycle 0
E = efficiency of amplification
Ct = threshold cycle
• If not, Ct values of the samples has to be
compared to a standard curve
Absolute quantification
Sample of Mel1 gene which had a Ct of 22.5 cycles after
amplification. What is the concentration of your amplicon?
Standard Curve
25.00
23.00
y = -3.1392x + 18.221
R² = 0.9993
Cycles
21.00
19.00
17.00
15.00
-1.6
-1.4
-1.2
-1
-0.8
-0.6
Log of DNA concentration
-0.4
-0.2
0
Absolute quantification
Concentration of Mel1 amplicon with Ct of 22.5
y = -3.1392 x + 18.221
22.5 = -3.1392 x + 18.221
x = -1.3630
10 -1.3630 (inverse Log 10)
The DNA concentration is 0.043 µg/ml
Relative quantification
• Normalization of the gene of interest to a
housekeeping gene
Sample
Ratio =
Housekeeping
Real-time PCR
Vs.
• More sensitive (need ~50
ng)
• More accurate (can
determine numbers)
• DNA template (stable)
• Doesn’t give size of
transcripts
• Faster (few hours)
• Requires less steps
• Less costly
Northern blotting
• Less sensitive (need ~10
ug)
• Less accurate (cannot
determine copy numbers)
• RNA template (unstable)
• Gives size of transcripts
• Long (hours to days)
• Requires numerous
processing steps
• More costly