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Subjects and Methods
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Subjects and Methods
The study is prospective case - control comparative study starting
from October 2014 to October 2015 in Molecular biology unit (MBU)
Faculty of Medicine (Benha University) and lab animal care centre
(LACC),Faculty of Veterinary medicine (Benha University) for measuring
the expression of DNA repair genes in normal immune cells in response to
cisplatin & oligonucleotide CpG-ODN treatment.
Subjects
Thirty two female Albino mice were included in this study. They
were classified into 4 main groups as follow:
Control group: Consisted of 8 mice. They injected by 200µl normal saline
treatment and balanced diet for 4 weeks.
Group I: Consisted of 8 mice. Animals of this group injected by CPGODN treatment at dose of 20 µg/mouse for 4 weeks
Group II: Consisted of 8 mice. Animals of this group injected by Cisplatin
treatment at dose of 3mg/kg.B.wt for 4 weeks.
Group III: Consisted of 8 mice. Animals of this group injected by
combination of Cisplatin and CPG- ODN treatment at dose of
(3mg/kg.B.wt + 20 µg/mouse) for 4 weeks.
1. Experimental animals:
Thirty two Albino mice were used in this study. They weighted (2025 g) and their age was around 4 weeks at the beginning of the
experiment. Mice were obtained from Lab animal care center, faculty of
Veterinary medicine (Benha University).
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Animals were kept for one week before experiment to acclimatize
to the laboratory conditions. The management was kept constant
throughout the experimental period. Water and normal balanced ration
was offered ad-libitum, and was re-newed everyday .Cages were cleaned
regularly in which mice were moved to completely clean cages two
times a week.
2. Cisplatin:
It was manufactured by MYLAN S.A.S Allee des Parcs SAINTPRIEST FRANCE as concentrated solution 1mg cisplatin /1 ml.
3. CpG-ODN phosphorothioated 1826:
It was manufactured by TRILINK BIOTECHNOLOGIES
COMPANY, San Diego,CA,USA
in lyophilized solid form.its
sequence 5\ TCC ATG ACG TTC CTG ACG TT 3\.
4. Solution and reagents used: RT2 RNA extraction kit (Qiagen,Gmbh) containing Buffer RLT
with (β- mercaptoethanol),Buffer RW1, Buffer RPE with 9699% ethanol, and RNase-Free Water.
 RT2 First Strand c DNA Kit (Qiagen,Gmbh) Buffer GE2
(gDNA elimination buffer) and BC5 Reverse Transcriptase Mix.
 RT2
SYBR
Green
qPCR
Mastermixes
(Qiagen,Gmbh)
containing HotStart DNA Taq Polymerase ,PCR Buffer , dNTP
mix (dATP, dCTP, dGTP, dTTP) ,SYBR Green dye and ROX
passive reference dye
 RT2 Profiler PCR Arrays (Qiagen,Gmbh) contains a primer for
each gene mixed with an inert dye.
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Methods:
Determination of DNA REPAIRING GENE Expression by PCR-array
Technique using ABI 7900 Real time machine:PCR Array System is the most reliable and accurate tool for
analyzing the expression of a focused panel of genes using SYBR
Green-based real-time PCR.
1. Tissue handling:Animals were sacrificed and biopsies of spleen were taken
immediately placed in ipendorf tube containing RNA latter and
stored at -80C for further processing.
2. Total RNA extraction:i. Tissue sample preparation:
 30 mg tissue sample collected in a micro centrifuge tube
disrupted and homogenized in 300 µl Buffer RLT using
a rotor–stator homogenizer.
 Add addition 200µl of lysis buffer to the homogenized
sample and vortex for 30 sec.
 Centrifuge at 10.000 g for 10 min.
 Carefully remove the supernatant by pipetting, and
transfer it to a new micro-centrifuge tube.
ii. Column loading
 Add 300µl isopropanol to the prepared cell lysate.
 Transfer the mixture directly into the Spin Column.
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 Centrifuge at 10.000g for 1 min. then discard the flowthrough.
iii. Primary column washing
 Apply 700µl of Primary Washing Buffer to the Spin
Column
 Centrifuge at 10.000g for 1 min. then discard the flowthrough.
iv. Secondary Column Washing
 Apply 700µl of Secondary Washing Buffer to the Spin
Column
 Centrifuge at 10.000g for 1 min. then discard the flowthrough
 Centrifuge again at 10.000g for 1min. to remove
residual ethanol.
v. Elution of RNA
 Place
the
Spin
Column
into
an
RNase-
free
microcentifuge tube
 Add 40µl Elution Buffer to the center of the column
membrane.
 Incubate at the room temperature for 1min. to elute the
RNA then store at -80 °C.
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3. RNA quantification and purity determined by Nanodrop
spectrophotometry
The concentration and purity of RNA s determined by measuring
the absorbance in the Nanodrop 2000 Spectrophotometer, thermoscientific. The spectral properties of nucleic acids are highly dependent
on pH. An absorbance reading of 1.0 at 260 nm in a 1 cm detection
path corresponds to an RNA concentration of 40 μg/ml.
 A260:A230 ratio should be greater than 1.7
 A260:A280 ratio should be 1.8 to 2.0
 Ratio should be 1.8 to 2.0 A260 should be >40 μg/ml.
4. Genomic DNA contamination
Eliminating genomic DNA contamination is essential for
obtaining optimal real-time gene expression profiling results using the
RT2 Profiler PCR Array. The genomic DNA control in each RT2
Profiler PCR Array specifically tests for genomic DNA contamination
in each sample during each run. A genomic DNA control threshold
cycle value of less than 35 indicates the presence of a detectable amount
of genomic DNA contamination that should be addressed.
5. cDNA Synthesis Using the RT2 First Strand Kit
a) The reagents of the RT2 First Strand Kit were centrifuged for 15
sec. to bring the content to the bottom of the tubes.
b) The genomic DNA elimination mix for each RNA sample was
prepared according to Table 1. Mixed gently by pipetting up and
down and then centrifuged briefly.
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Table 1: Genomic DNA elimination mix:
Component
Amount
RNA
5 μg
Buffer GE
2 μl
variable
RNase- free water
10 μl
Total volume
c) The genomic DNA elimination mix was incubated for 5 min at
42°C, then placed immediately on ice for at least 1 min.
d) The reverse-transcription mix was prepared according to Table 2.
Table 2: Reverse-transcription mix:
Component
Volume for 4 reactions
5x Buffer BC3
16 μl
Control P2
4 μl
RE3 Reverse Transcriptase Mix
8 μl
RNase-free water
12 μl
Total volume
40 μl
e) 10 μl of reverse-transcription mix was added to each tube
containing 10 μl genomic DNA elimination mix then mixed
gently by pipetting up and down.
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f) The mix was incubated at 42°C for exactly 15 min. Then
immediately the reaction was stopped by incubating at 95°C for 5
min.
g) 91 μl RNase-free water was added to each reaction. Mixed by
pipetting up and down several times.
h) The reactions were transferred and stored at –20°C freezer.
6. Real-Time PCR for RT2 Profiler PCR Array
a) The RT2 SYBR Green Mastermix was briefly centrifuged for
(10–15 s) to bring the contents to the bottom of the tube.
b) The PCR components mix was prepared in a loading reservoir
depending on the RT2 Profiler PCR Array format, as described in
Table 3.
N.B: the remaining 9 μl cDNA synthesis reaction was saved at 20°C, as it may be needed to perform quality control analysis.
Table 3: PCR components mix
Array format
96-well
2x RT2 SYBR Green Mastermix
1350 μl
cDNA synthesis reaction
102 μl
RNase-free water
1248 μl
Total volume
2700 μl
c) Dispense the PCR components mix into the RT 2 Profiler PCR
Array.
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 The RT2 Profiler PCR Array was carefully removed from its
sealed bag (figure 11, 12).
Figure 11: Design of RT2 Profiler PCR Arrays
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Figure 12(a): Description of the genes included in RT2 Profiler PCR Arrays.
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Figure 12(b): Description of the genes included in RT2 Profiler PCR Arrays.
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Figure 12(c): Description of the genes included in RT2 Profiler PCR Arrays.
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Figure 12(d): Description of the genes included in RT2 Profiler PCR Arrays.
 25 μl PCR components mix was added to each well of the RT2
Profiler PCR Array.
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d) Carefully, the RT2 Profiler PCR Array was tightly sealed with
Optical Thin-Wall 8-Cap Strips.
e) Centrifuged for 1 min at 1000 g at room temperature (15–25°C)
to remove bubbles. The plate from underneath was visually
inspected to ensure no bubbles were present in the wells as the
presence of bubbles in the wells interferes with results.
f) The RT2 Profiler PCR Array was placed on ice while setting up
the PCR cycling (ABI 7900 Real time machine) program.
g) The real-time cycler was programmed according to Table 4 then
the RT2 Profiler PCR Array was placed in it.
Table 4: Cycling conditions.
Cycles
Duration
Temperature
1
10 min
95°C
Comments
HotStart
DNA
Taq
Polymerase is activated by
this heating step.
40
15 sec
95°C
1min
60°C
Perform fluorescence data
collection.
Data Analysis:
 Calculate the threshold cycle (ΔCT) for each well using the
real-time cycler software.
 Export the CT values for all wells to a blank Excel®
spreadsheet for use with the SABiosciences PCR Array Data
Analysis Template Excel or Web-based software.
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
Dissociation (melting) curve analysis was performed to verify
PCR specificity.
 A melting curve program was run and generated a first
derivative dissociation curve for each well using the real-time
cycler software.
 The fold-change for each gene calculated using the formula:
2(-ΔΔCT) (Livak and Schmittgen, 2001).
The p values are calculated based on a Student’s t-test of the replicate 2(ΔCt) values for each gene in the control group and treatment groups.
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