m Applications in Oncology
Quantification of
MORl Expression in
Different Cell Lines
Primer and Probe
Design
Optimization of
Assay
6.5
6
Y = 11.9 - 0.27 * X. RA2 = .997
'" 5.5
~
."
a. 5
E
~ 4.5
a
:!:.
4
C\
0
....J
3.5
3
2.5
20
22
24
26
28
30
32
34
Thresho ld cycle number
Fig. 2. The quantification curve was established by plotting the log of the copy number of templates used for amplification versus the threshold cycle number. The results showed a linear
inverse correlation (R2=0.998, P
reaction. The standard curve established above was then used to calculate the
copy number of MDRI mRNA molecules in each sample by extrapolation. The
results indicated that a single cell from the resistant cell line contained 13 copies
of MDRI mRNA and a single cell from the sensitive cell line was determined to
contain one copy of MDRI mRNA. Therefore, there is a 13-fold difference in the
expression of the MDRI gene between the chemotherapy agent-resistant cell line
and the sensitive cell line.
Comment
The primers should have the T mS as close to each other as possible for the best
amplification efficacy. The hybridization probe should have a T m about 5-10 0 C
above that of primers to favor probe binding over primer binding.
The most important factors for optimizing the assay are Mg++ concentration and
the concentration of each primer and hybridization probe. Different primer ratios
and different probe concentrations change the signal intensity generated from the
PCR products (data not shown). Multistep annealing during the melting curve
analysis enhances the signal intensity compared with one-step annealing (data
not shown). Monitoring the ratio of the acceptor channel (F2) fluorescence intensity to fluorescein (Fl) provides a smoother quantification curve than monitoring the F2 alone.
Quantification of
MORl Expression in
Different Cell Lines
Primer and Probe
Design
Optimization of
Assay
6.5
6
Y = 11.9 - 0.27 * X. RA2 = .997
'" 5.5
~
."
a. 5
E
~ 4.5
a
:!:.
4
C\
0
....J
3.5
3
2.5
20
22
24
26
28
30
32
34
Thresho ld cycle number
Fig. 2. The quantification curve was established by plotting the log of the copy number of templates used for amplification versus the threshold cycle number. The results showed a linear
inverse correlation (R2=0.998, P
reaction. The standard curve established above was then used to calculate the
copy number of MDRI mRNA molecules in each sample by extrapolation. The
results indicated that a single cell from the resistant cell line contained 13 copies
of MDRI mRNA and a single cell from the sensitive cell line was determined to
contain one copy of MDRI mRNA. Therefore, there is a 13-fold difference in the
expression of the MDRI gene between the chemotherapy agent-resistant cell line
and the sensitive cell line.
Comment
The primers should have the T mS as close to each other as possible for the best
amplification efficacy. The hybridization probe should have a T m about 5-10 0 C
above that of primers to favor probe binding over primer binding.
The most important factors for optimizing the assay are Mg++ concentration and
the concentration of each primer and hybridization probe. Different primer ratios
and different probe concentrations change the signal intensity generated from the
PCR products (data not shown). Multistep annealing during the melting curve
analysis enhances the signal intensity compared with one-step annealing (data
not shown). Monitoring the ratio of the acceptor channel (F2) fluorescence intensity to fluorescein (Fl) provides a smoother quantification curve than monitoring the F2 alone.