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Chapter 3
METHODOLOGY
Chemicals and reagents
The various chemicals and molecular biology reagents were procured from standard
sources as described in appendix I. The composition and preparation of reagents are given
in appendix II.
Specimen
Patients and Controls
This study included 150 primary gastric cancer patients. All blood and tissue with
adjacent normal samples for this study were taken from gastric cancer patients registerd in
the Department of Surgical Gastroenterology, Sher-I-Kashmir Institute of Medical Sciences,
from March 2009 to July 2012. Tumor types and stages were determined by two
experienced pathologists. Blood samples of 250 age, gender, dwelling and smoking matched
cases with no signs of any malignancy or any other disease were collected for controls. Data
on all gastric cancer patients were obtained from personal interviews with patients and or
guardians, medical records and pathology reports. The data collected included gender, age,
dwelling, tumor location, lymph node status, site of growth, EGD biopsy and salt tea
consumption. All patients and or guardians were informed about the study, and their consent
to participate in this study was obtained on a predesigned questionnaire (available on
request). The collection and use of tumor and blood samples for this study was approved by
the appropriate Institutional Ethics Committee.
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Sample collection/storage
1ml of peripheral blood was obtained from each subject in sterile 1.5ml microfuge
tubes containing 10µl of 0.5M EDTA (pH 8.0) as an anticoagulant and stored at -20°C till
use. Approximately 500mg of surgically resected tumour tissue and adjacent normal tissue
at a distance of 10 cm from the tumour site were collected directly into sterile vials
containing chilled PBS (pH 7.4)/Formalin embedded frozen tissues and frozen at -70°C for
molecular investigations. Histopathological report of the collected tumour tissues was
obtained from the Department of Pathology of the SKIMS. Histopathologicaly confirmed
gastric
cancer
tissues
and
corresponding
normal
tissues
were
used
for
immunohistochemistry and semiqauntative reverse transcriptase PCR of IL-1 and IL-8 gene,
while blood samples were used for polymorphic analysis of IL-1, IL-8 and IL-10 gene.
Extraction and Quantitation of genomic DNA
Principle
DNA is extracted from the mammalian cells by lysing the cell membranes
using detergents like SDS. The protein content of the cells is then precipitated either by
organic solvents like phenol-chloroform-isoamyl mixture or by various salts such as sodium
chloride, ammonium acetate or potassium acetate. Finally the DNA is preicipated by ethanol
or isopropanol and the DNA pellet is dissolved in Tris- EDTA.
DNA was extracted from the tissues by Phenol - Chloroform method and by Qiagen
DNA extraction kit while Salting out method was used for the extraction of DNA from
blood samples.
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Phenol - Chloroform method (Blin et al., 1976)
The frozen tissue was allowed to thaw at room temperature.
 The tissue was chopped with fresh surgical blades in a sterile petri dish.
 The chopped tissue was then transferred into a sterile polypropylene tube (15ml)
containing 3ml of 1X TE, 2ml of lysis buffer and proteinase K (100μg/ml) was
added to it.
 The mixture was incubated at 37°C in a water bath for overnight.
 Next day, equal volume of TE saturated phenol (pH 8.0) was added and the mixture
was gently mixed by inversion of tubes on overhead shaker for 15 minutes.
 The tubes were then centrifuged at 3000-4000rpm at 4°C for 15 minutes.
 The supernatant was transferred to a fresh polypropylene tube without disturbing the
interphase with the help of a micropipette fitted with a wide bored tip.
 To the supernatant, equal volume of TE saturated phenol - chloroformisoamylalcohol (25:24:1) was added and the mixture was shaken on overhead shaker
for 15 minutes and steps 6 and 7 repeated.
 To the supernatant thus obtained in fresh tube, equal volume of chloroform isoamylalcohol (24:1) was added and each tube was shaken, and step 6 and 7
repeated.
 To the supernatant from the above step, 1/10 volume of chilled 3M sodium acetate
solution (pH 5.2) and 2.5 volumes of chilled ethanol or equal volume of isopropanol
was added and mixed by gently inverting the tube. If visible precipitate of genomic
DNA appeared, it was transferred to 1.5ml microfuge tube and centrifuged at
6000rpm for 5 minutes. The pellet thus obtained was washed with 500μl of 70%
ethanol and re-centrifuged. The washing was repeated.
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 If the precipitate of genomic DNA was not visible, the tubes were then allowed to
stand at either -70°C for 10 minutes or at -20°C for overnight. Next day, the tube
was centrifuged at 6000 rpm at 4°C for 45 minutes. The pellet thus obtained was
washed twice with 70% ethanol as above.
 Air dried DNA pellet was dissolved in 200μl of TE (pH 7.6) and stored at 4°C or at 20°C for longer periods.
Salting out method
 300μl aliquots of whole blood were transferred to each of two microfuge tubes.
 900μl of 20mM Tris-Cl (pH 7.6) was added to each tube and inverted to mix the
contents.
 The tubes were incubated at room temperature for 10 minutes, occasionally inverting
the tubes.
 The tubes were centrifuged at maximum speed for 20 seconds at room temperature
in a microfuge.
 All of the supernatant was discarded except 20μl and the pellets of white cells were
resuspended in the small amount of supernatant left in each tube. The resuspended
cell pellets were combined in a single tube.
 To the resuspended white blood cell pellets, 600μl of ice-cold cell lysis buffer, 3μl of
proteinase K solution was added and incubated for at least 3 hours but no more than
16 hours at 55°C.
 The digests were allowed to cool to room temperature. 200μl of potassium acetate
solution was added to each tube and the contents of the tube were mixed by
vortexing vigorously for 20 seconds.
 The precipitated protein/SDS complex was pelleted by centrifugation at maximum
speed for 3 minutes at 4°C in a microfuge.
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 The supernatant was transferred to a fresh microfuge tube containing 600μl of
isopropanol. The solution was mixed well and the precipitate of DNA was recovered
by centrifuging the tube at maximum speed for 1 minute at room temperature in a
microfuge.
 The supernatant was removed and 600μl of 70% ethanol was added to the DNA
pellet. After inverting the tube several times, it was centrifuged at maximum speed
for 1 minute at room temperature in a microfuge.
 The supernatant was removed carefully and the DNA pellet was allowed to dry in air
for 15 minutes.
 The pellet of DNA was redissolved in 100μl of TE (pH 7.6).
Quantitation
The concentration of the extracted DNA was measured in a spectrophotometer at 260nm
wavelength by using the formula:
DNA μg/ml = A260 x 50 X dilution factor.
The purity of DNA was checked by using A260/ A280 ratio.
Agarose gel electrophoresis (AGE)
The quality of the DNA obtained from the tissue specimens and blood samples was
analyzed on 1% agarose gel.
Principle
Electrophoresis is a technique for separation of molecules based on molecular
weight and electrical charge. The matrix used in AGE is agarose with ethidium bromide dye
added to it. Agarose is a linear polysaccharide obtained from seaweed and is made up of the
basic repeat unit- agarobiose, which comprises of alternating units of galactose and 3, 6 anhydrogalactose. Ethidium bromide dye, which is added in the gel, gets intercalated
between the bases of the DNA thereby converting the twisted helix to a linear structure.
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Upon electrophoresis with Tris-borate/acetate buffer serving as electrolyte, negatively
charged DNA in the gel moves towards positive electrode (anode).
The mobility of DNA depends upon
 Concentration of agarose in the gel - As the concentration of the agarose increases,
the resolving power also increases and the mobility of the DNA is slower.
o Mobility of DNA 1/α Concentration of Gel
 Molecular weight of DNA - Smaller fragments move faster than the larger ones.
o Mobility of DNA 1/ α Molecular weight.
Procedure
 The edges of the gel running tray were sealed with tape to form a mould. The tray
was set on a leveled bench and an appropriate comb for forming the sample slots in
the gel was positioned 0.5-1.0mm above the plate to form a complete well when
agarose was poured in the mould.
 To 0.3g of agarose 30ml of 1X TAE buffer was added in a conical flask, and the
level of the slurry was marked with a marker on the flask. The volume was sufficient
to prepare a gel of 3mm-5mm thickness.
 The slurry was heated in a boiling-water bath until the agarose dissolved. The
volume of the solution was checked with the help of the mark made on the flask and
was replenished with water when needed. The gel solution was allowed to cool
down to about 50°C and ethidium bromide was added to a final concentration of
0.5μg/ml. The gel solution was mixed thoroughly by gentle swirling.
 The warm agarose was poured into the sealed gel tray and allowed to set completely
for 30-45 minutes at room temperature. When the gel was well set, the comb and the
tape was carefully removed and the gel was mounted in the electrophoresis tank.
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Electrophoresis buffer (1X TAE) was added to the tank to cover the gel to a depth of
about 1-2 mm.
 2μl of DNA samples, 3μl of 6X gel loading buffer were mixed in microfuge tubes
and loaded into the wells along with a molecular size marker on one side of the gel.
 Electrophoresis was carried out at 80 volts until dye had migrated a sufficient
distance through the gel. The electric current was turned off and the gel was
examined on a UV illuminator and photographed.
 The high-molecular-weight DNA was used for further molecular
investigation.
Polymerase chain reaction
Principle and application
Polymerase chain reaction (PCR), a powerful technique developed by a team headed
by Kary Mullis at Cetus Corporation, is used to amplify a segment of DNA in vitro (Mullis
et al., 1987). This method can produce large amount of a specific DNA sequence from a
complex DNA template in a simple enzymatic reaction. This method utilizes a DNA
polymerase and two oligonucleotide primers to synthesize a specific DNA from a single
stranded template sequence. The oligonucleotides typically have different sequences and are
complementary to sequences that lie on opposite strands of the template DNA and flank the
segment of DNA that is to be amplified. The length of the primers usually 20 bases or more
must be sufficient to overcome the statistical likelihood that their sequence would occur
randomly in the overwhelmingly large number of non-target DNA sequences in the sample.
PCR is carried out in a series of cycles. Each cycle begins with a denaturation step to render
the target DNA single stranded. This is followed by an annealing step during which the
primers anneal to their complementary sequences so that their 3' hydroxyl ends face the
target. Finally each primer is extended through the target region by the action of DNA
polymerase. Since the products of one round of amplification serve as templates for the next
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cycle, the three step cycles are repeated until a sufficient amount of the product is produced.
The major product of this exponential reaction is a segment of double-stranded DNA whose
termini are defined by the 5' termini of the oligonucleotide primers and whose length is
defined by the distance between the primers. In addition, longer DNA molecules are
generated during the reaction. For example, the products of a successful first round of
amplification are heterogeneously sized DNA molecules, whose lengths may exceed the
distance between the binding sites of the two primers. In the second round these molecules
generate DNA molecules of defined length that will accumulate in an exponential fashion in
the later rounds of amplification and will form the dominant products of the reaction.
Although longer molecules continue to be formed from the original DNA template in every
round, they accumulate only at a linear rate and therefore do not contribute significantly to
the final product.
The earliest PCR experiments utilised the Klenow fragment of Escherichia coli
DNA polymerase I at a temperature of 37°C and often produced incompletely pure target
products as judged by gel electrophoresis. However, the isolation of a heat-resistant DNA
polymerase from Thermus-aquaticus (Taq) (Chien et al., 1976) allows primer annealing
and extension to be carried out at an elevated temperature, thereby reducing mismatched
annealing to non-target sequences. This added selectivity results in the production of large
amounts of virtually pure target DNA. Another important advantage of Taq polymerase is
that it escapes inactivation at higher temperatures and need not be replaced after every
denaturation step. This has allowed automation of PCR using machines that have controlled
heating and cooling capability. This results in substantial improvements in the specificity
and yield of the amplification reactions and the size of the amplified product. For example,
between 0.5μg and 1.0μg of target DNA up to 2kb in length can be obtained from 30-35
cycles of amplification with only 6-10μg of genomic DNA.
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Procedure for PCR amplification of IL-1, IL-8 and IL-10 gene
All precautions were taken to ensure contamination free amplification of DNA.
Prevention of carry-over by separation of pre and post-PCR steps and use of aerosol-free
tips were strictly followed. Results were considered valid only on reproduction of same in
two independent experiments.
Equipments and Reagents
 Thermal cycler (Bio-rad Thermocycler)
 Micropipettes
 0.2ml PCR tubes
 Genomic DNA: 250ng/μl
 10X PCR buffer: 100mM Tris-HCL, pH 8.3; 500mM KCL; 15mM MgCl2;
0.1% gelatine and 1% TritonX 100
 Deoxyribonucleotide triphosphate: 10mM each dATP, dCTP, dGTP and
dTTP
 Primers: 10μM in sterile double distilled water
 Taq DNA polymerase: 5U/μl
Primers for amplification
Primers for the amplification of IL-1B, IL-8, IL-10 gene polymorphisms were was
obtained from the literature Table:1.
Protocol
The amplification reaction for sequencing was carried out in 50μl reaction volume in
0.2ml PCR tubes while for genotyping and SSCP, the reaction volume was 25μl. The
reaction contained the following reagent volumes: 10X PCR buffer
 10mM dNTP mix
 Sense Primer
2.5μl
0.5μl
0.5μl
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




Antisense Primer
Taq DNA polymerase (5U/μl)
Genomic DNA
Distilled water
Total volume
0.5μl
0.125μl
1.0μl
19.875μl
25.0μl
The above mentioned reagents were pipetted in a 0.2ml thin walled PCR tube and
placed in Thermocycler. The following temperature profile was used for amplification:




Initial denaturation
Denaturation
Annealing
Extension
Final Extension
95°C for 5minutes
95°C for 35 seconds
x°C for 35 seconds*
72°C for 50 seconds
72°C for 7 minutes
Temperature profile from step 2-4 was used for 35 cycles before final extension. * x
was 3-5°C lower than the melting temperature (Tm) of the primers and was calculated by
using the following formulae.
For primers 14-25 nucleotides in length:
Tm = [2°C x (number of A and T bases)] + [4°C x (number of G and C bases
The amplified products were detected and confirmed by comparing with
a 100bp DNA marker ladder by electrophoresis on 2% agarose gel containing
ethidium bromide (0.5μg/ml) in a mini gel system, as described in the section on agarose
gel electrophoresis with few changes which are listed below: To 0.6g of agarose, 30ml of 1X TAE buffer was added in a conical flask and placed in
boiling water bath until agarose was completely dissolved.
 After placing the well set gel with buffer in the electrophoresis unit, 10μl of PCR
products mixed with 2μl of 6X gel loading buffer was loaded into the wells along with
100bp DNA marker ladder.
 Electrophoresis was carried out at 100 volts until dye had migrated a sufficient distance
through the gel. The electric current was turned off and the gel was examined on a UV
illuminator and photographed.
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RFLP
Principle
The restriction endonuclease type II for SNP detection is selected, such that it
recognizes and cleaves one of the polymorphic bases. Upon incubation at optimum
temperature and for optimum time with a buffer, the enzyme restricts the DNA, at a specific
site. Electrophoresis of the digested products yields fragments of sizes based on the
cleavage pattern. If both the alleles harbor the base recognized by the enzyme, fragments of
sizes accounting cumulative to the undigested product are obtained (homozygous for that
base). If one of the allele harbors a different base, then the single allele is cleaved resulting
usually in 3 fragments- the original undigested product, and two digested fragments of sizes
cumulatively accounting to the original PCR product (Heterozygous). Absence of the base
recognized by the enzyme does not result in digestion thereby retaining the original PCR
product (Homozygous).
Restriction enzymes for RFLP
Restriction enzymes for the Interlukins were obtained from the literature.
Restriction digestion
10µl of the PCR products were subjected to restriction digestion respectively. The
reaction conditions were set up according to the supplier of restriction enzymes.
The PCR products were visualized on a 3% agarose gel containing 0.5 µg/ml
ethidium bromide and photographed. The genotypes of >20% of the samples were double
blindly reassessed to confirm the results by two independent researchers.
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Automated DNA sequencing
Purified PCR products showing digestion by RFLP analysis as well as randomly
chosen samples were used for direct DNA sequencing using the ABI prism 310 automated
DNA sequencer. To minimize the sequencing artifacts by PCR, products from at least two
different PCRs were sequenced using both forward and reverse primers.
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Amplicon
Primer sequence*
1) 1) 5’-AGA AGC TTC CAC CAA TAC TC-3’
IL-1B-31
Annealing Restriction
Product
Temp.(°C) enzymes
size (bp)
57
AluI
239
57
AvaI
304
60
MunI
798
61
VspI
533
58
MnII
218
59
RsaI
542
2) 2) 5-AGC ACC TAG TTG TAA GGA AG-3′
IL-1B-511
1) 5-TGG CAT TGA TCT GGT TCA TC-3
2) 5-GTT TAG GAA TCT TCC CAC TT-3
1) 5’- CAC TGG AAT TAA TGT CTT AGT ACC A -3’
IL-8-251
2) 5- AAG CTT GTG TGC TCT GCT GTC TCT -3′
IL-8-845
1) 5-AAC CAA GCA GCT CCA GTG-3
2) 5-AAT GAT TGG CTG GCT TAT CT-3
IL-10-1082
1) 5-TCT GAA GAA GTC CTG ATG TC-3
2) 5-CTC TTA CCT ATC CCT ACT TCC-3
IL-10-592
1) 5-GAC TCC AGC CAC AGA AGC TTA-3
2) 5-ATA TCC TCA AAG TTC CCA AGC-3
* 1) Sense primer, 2) Antisense primer
Table;
1
Primers
used
for
amplification
of
different
interlukins
gene
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Immunohistochemistry
Immunostaining methods
Immunostaining was done by the method of (Ahmad et al., 2011). Sections of
formalin-fixed, paraffin-embedded Gastric Tissue (Tumor and Adjacent Normal) were
obtained on poly-l-lysine coated slides. Sections were deparaffinized in xylene, then
rehydrated through a graded alcohol series. Antigen retrieval was performed by incubating
slides in citrate buffer (pH 6.0) (10 mM) at 95 ◦C for 20 min. Endogenous peroxidase activity
was blocked with 3% H2O2 for 30 min. To detect IL-1 and IL-8 immunoreactivities, sections
were incubated under humid conditions overnight at 4 ◦C with the following monoclonal
antibodies: anti IL-1 and anti-IL-8 antibody (1;400 and 1:200; Thermo Fisher Scientific,
USA). Next day, the slides were washed three times in Tris buffers (pH 6.0) and were
incubated with a biotinylated Human Anti-Polyvalent Plus (Thermo Fisher Scientific, USA)
for 30 min at room temperature. This step was followed by further wash in Tris buffer and
incubation of slides at room temperature with a Streptavidin Peroxidase Plus (Thermo Fisher
Scientific, USA) that binds to the biotin present on the secondary antibody. After washing in
Tris buffer, the immunostaining reaction product was developed using 3, 3-diaminobenzidine
(DAB Plus substrate, Thermo Fisher Scientific, USA). After immunoreactivity, slides were
dipped in distilled water, counterstained with Harris hematoxylin and finally the sections
were dehydrated in xylene, mounted with DPX and coverslipped. Negative controls included
staining tissue sections with omission of the primary antibody whereas positive control slides
were also run in parallel in each case. Slides prepared for each case were examined by light
microscopy.
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Quantitative evaluation of IL-1 and IL-8
Semi-Quantitative intensity scale ranging from 0 for no staining and +3 for maximum
staining is used for calculating Immunohistochemical score. According to the diffuseness of
the DAB staining, sections were graded as 0 (no staining), 1 (staining, 25%), 2 (staining
between 25% and 50%), 3 (staining between 50% and 75%), or 4 (staining >75%).
According to staining intensity, sections were graded as follows: 0 (no staining), 1 (weak but
detectable staining), 2 (distinct staining) or 3 (intense staining). Immunohistochemical
staining scores were obtained by adding the diffuseness and intensity scores. All slides were
examined by two independent observers who were unaware of the experimental protocol.
The slides with discrepant evaluations were reevaluated, and a consensus was reached.
Measurements were carried out using an Olympus BX51 (Hamburg, Germany) microscope
using objectives with 10× and 40× magnifications.
Semiquantitative Reverse Transcription-PCR (RT-PCR)
Total RNA was extracted after homogenisation with 1 ml/100 mg of gastric tissues by
TRIzol (Invitrogen Life Technologies, USA). RNA extracted, quantity and quality was
analyzed by UV spectrophotometer and agarose gel electrophoresis. One microgram total
RNA was reverse transcribed using RevertAidTM first Strand cDNA Synthesis Kit
(Fermentas, Life Sciences, USA). By use of first-strand cDNA as a template, the specific
primers for IL-1β sense 5-CAGTGAAATGATGGCTTATTAC-3 and antisense 5CTTTCAACACGCAGGACAGGT-3 primers yield a 548-bp product and specific primers
for
IL-8
sense
5-AAGGAACCATCTCACTG-3
TTCTTGGATACCACAGAG-3
and
antisense
5-
primers yield a 352-bp product and GAPDH sense 5-
CAAGGTCATCCATGACAACTTTG-3and antisense 5-TCCACCACCCTGTTGCTGTAG3 primers yield 496 bp (as reference gene) were subjected to 35 cycles of PCR amplification
(30 s denaturation at 94 ◦C, 30 s annealing temperature (IL-1 550c and IL-8
700c and
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GAPDH 580c, 2 min extension at 72 ◦C) in a thermal cycler. The amplified products were
resolved by gel electrophoresis on 1.5% agarose and visualized by ethidium bromide
(0.5µg/ml). Images of the RT-PCR ethidium bromide stained agarose gel were acquired
using AlphaImagerTM Gel Documentation, USA. Quantification of the results was
accomplished by measuring the optical density of the cDNA bands. The intensities of bands
of above mentioned genes were normalized relative to that of GAPDH bands by dividing the
former by the GAPDH specific PCR product densities. GAPDH acted as control for sample
to sample variations in reverse transcription and PCR conditions and the extent of
degradation and recovery of RNA.
Statistical analysis
Statistical analyses were performed with the SPSS version 16 software. Observed
frequencies of genotypes in gastric cancer patients were compared to controls using chisquare or Fisher exact tests when expected frequencies were small. The chi-square test was
used to verify whether genotype distributions were in Hardy-Weinberg equilibrium.
Statistical significance was set at P < 0.05. Odds ratio at 95% confidence intervals (95% CI)
and P values were computed by binary logistic regression, and all results were adjusted for
age, gender, salt tea consumption, smoking, and dwelling. The independent sample
Student’s t-test was applied to check association between cases and controls. The P values <
0.05 were considered to indicate statistical significance in these tests. All analyses were
performed using the statistical package SPSS ver. 16 (SPSS Inc., Chicago, IL).Haplotype
analysis was performed by Shesis online version.
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