Chapter two Materials and Methods

Chapter two

Materials and Methods

2-1 Materials 2.1.1 Equipments and apparatus The instruments and their manufactures used in the present study are summarized in the following table. Table 2.1 List equipments used in the genomic analysis with their companies and origins

No.

Instrument

Company

Origin

1.

Real-time polymerase chain reaction

Cepheid

US

(thermal cycler) 2.

Gel electrophoresis

Thermo Scientific

US

3.

Epindorff tubes

Epindorff

Germany

4.

Water bath

Market

China

5.

Digital camera

Canon

Japan

6.

Microcentrifuge

Thermo Scientific

US

7.

Incubator

Carbolite

England

8.

spin centrifuge

Benchmark

US

9.

micropipettes Pipettes (adjustable)

Epindorff

Germany

10.

microwave oven

Nawal

Turkey

11.

Refrigerator, Freezer

Concord

France

12.

UV transilluminator

Major Science

Taiwan

13.

UV-visible spectrophotometer and

T80-vis/UV

UK

Stuart Scientific

UK

Quartz cuvettes 14.

Vortex 45

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Materials and Methods

2.1.2 Materials Table 2.2 List of the materials used in the genomic analysis with their companies and origins

No.

Chemicals

Company

Origin

1.

Agarose

Promega

USA

2.

Nuclease free water

Promega

USA

3.

Ethanol (75% and 100%)

BDH

England

4.

ethidium bromide solution 10mg/ml)

Promega

USA

5.

blue/orange loading dye 6x

Promega

USA

6.

proteinase K (PK)

QIAGEN

Germany

7.

TAE 40x

Promega

USA

8.

Xylene

Fluka

Switzerland

9.

MinElute Columns

QIAGEN

Germany

10.

Buffers, Collection Tubes (2 ml)

QIAGEN

Germany

11.

RNA ase

QIAGEN

Germany

Promega

USA

12.

DNA Ladder (100bp), DNA Ladder (1kb)

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2.1.3 Kits used for molecular tests: Table 2.3 the kits used in the experiments.

No. 1.

Kits

Company

HPV High Risk Screen Real- TM Sacace, Biotechnologies, Caserta, Quant 2X

Italy. Catalog No. TV31-100/2FRT 2x

2.

Real Time PCR kit for qualitative Sacace, Biotechnologies, Caserta, detection of Human Papilloma Italy virus 6 and 11

3.

Catalog No.TV11-100FRT

QIAamp DNA FFPE Tissue Kit QIAGEN, Germany (50)

Catalog no. 56404,Number of preps 50

2.2

Study patients:-

2.2.1 Study group: The study was carried out on 120 females (100 patients and 20 controls); ages (range from19- 62 years) were included and studied during the period from December 2014 to April 2015. They included 80(100%) patients who were diagnosed at private clinic in Ramadi from patients with different cervical lesions and 20 (100%) patients. These were

represented

by

formalin-fixed,

paraffin

embedded

tissue,

(retrospective study samples) from patients with different cervical Cancer. A Questionnaire form was designed for data collection from recruited individuals included in this study and all the relevant obstetric and gynecological findings were and control group (health individuals including natural and regular of menstrual cycle, pH 3.8-4.5, no itching, 47

Chapter two

Materials and Methods

health discharge doesn’t have a strong smell and color, no irritation and no infection) (the Appendix).

2.2.2 Sample collection, Storage and Transportation Cervical swabs: A- Excess mucus was removed from the cervical os and surrounding ectocervix used a cotton or polyester swab. This swab was discarded. B- The sample cervical brush was inserted 1.0-1.5 centimeters into the cervical os until the largest bristles touch the ectocervix which has not inserted brush completely into the cervical canal. Rotate brush 3 full turns in a counterclockwise direction, removed from the canal. C- The brush was inserted into the nuclease-free 2.0 ml tube with 300 µL of transport medium (Sacace). D- The brush was vigorously agitated in medium for 15-20 sec. E- Snap off shaft at scored line, the brush end was leaved inside tube. The cervical swab was processed immediately in the laboratory by centrifugation at 3500 rpm for 10 minutes and the supernatants stored at – 20ᵒC until analyzing them at the laboratory ASCo. Learning Center, Baghdad, Iraq (Sambrook et al., 2004).

2.3 Molecular methods 2.3.1 DNA Extraction from cervical lesion swabs for the detection the Human Papilloma Virus genotypes 6 and 11 1. Lysis and washing solutions (in case of their storage at 2-8°C) should be warmed up to 60–65°C until the disappearance of ice crystals. The required quantity of 1.5 ml polypropylene tubes was prepared included 48

Chapter two

Materials and Methods

one tube for negative control of extraction. 2. To each tube 300 μl of lysis solution, was added. 3. 100 μl of samples was added to the appropriate tube. 4. The controls were prepared as follows: -100 μl of C– (Neg Control provided with the amplification kit) was added to the tube and labeled C neg. 5. The tubes were vortexed and incubated for 5 min at 65°C for 7-10 sec and centrifugated. If the sample was not completely dissolved recentrifuge of the tube for 5 min at a maximum speed (12000-16000 g.) was occurred and transferred the supernatant into a new tube for DNA extraction. 6. Sorbent was vortexed vigorously and 20 μl to each tube was added. 7. Vortexing was achieved for 5-7 sec and all tubes were incubated for 3 min at room temperature. This step was repeated. 8. All tubes were centrifugated for 30 sec at 5000g and a micropipette was used with a plugged aerosol barrier tip, carefully removed and discarded supernatant from each tube without disturbing the pellet. Changed tips are between the tubes. 9. 500 μl of washing solution was added to each tube and vortexed vigorously and centrifugated for 30 sec at 10000g. Removed and discarded supernatant was removed and discarded from each tube. 10. Step 9 was repeated and incubated all tubes were incubated with open cap for 5-10 min at 65°C. 11. The pellet was resuspended in 100 μl of DNA- eluent and incubated for 5 min at 65°C and vortexed periodically. 49

Chapter two

Materials and Methods

12. The tubes were centrifuged, for 1 min at 12000g. 13. The supernatant, contained DNA ready for amplification. If amplification was not performed in the same day of extraction, the processed samples can be stored at 2-8°C for at maximum period of 5 days or frozen at -20°/ -80°C (Sambrook et al., 2004; Aumran, 2000). 2.3.2 Protocol 1. The required quantity of reaction tubes for samples (N) and controls (N+2) were prepared. 2. The mixture was prepared as follows for 60 samples: PCR- buffer-FRT 30 μl of TaqF DNA Polymerase was added into the tube. The tube was vortexed carefully. This mix was stabled for 3 months at +4°C. 3. Reaction mix was prepared by adding for each sample into the new sterile tube 10 μl of PCR-mix-1-FRT and 5 μl of mix PCR- buffer-FRT/ Taq F DNA Polymerase (see table 2.4). 4. To each reaction tube was added 15 μl of reaction mix which was added and 10 μl of extracted DNA and mixed by a pipette. 5. For each panel 2 controls were prepared: ● Ten μl of DNA-buffer was added to the tube labeled amplification negative control; ● Ten μl of Positive Control Complex C+ was added to the tube labeled amplification positive control; 6. The tubes were inserted in the thermal cycler. The results were interpreted through the presence of crossing of fluorescence curve with the threshold line.

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Table 2.4 Pipetting scheme for the quantity of reagents for N samples sample

5

6

7

8

9

10

11

12

13

14

15

16

17

18

19

20

21

PCR-mix-1-FRT

80

90

100

110

120

130

140

150

160

170

180

190

200

210

220

230

240

PCR-buffer-

40

45

50

55

60

65

70

75

80

85

90

95

100

105

110

115

120

sample

22

23

24

25

26

27

28

29

30

31

32

33

34

35

36

37

38

PCR-mix-1-FRT

250

260

270

280

290

300

310

320

330

340

350

360

370

380

390

400

410

PCR-buffer-

125

130

135

140

145

150

155

160

165

170

175

180

185

190

195

200

205

sample

39

40

41

42

43

44

45

46

47

48

49

50

51

52

53

54

55

PCR-mix-1-FRT

420

430

440

450

460

470

480

490

500

510

520

530

540

550

560

570

580

PCR-buffer-

210

215

220

225

230

235

240

245

250

255

260

265

270

275

280

285

290

FRT/TaqF

DNA

Polymerase

FRT/TaqF

DNA

Polymerase

FRT/TaqF

DNA

Polymerase

Note: the calculation of the quantity of mixes

2.3.3 DNA quantitation: Ten µl of DNA sample was added to 990µl of distilled water, and mixed thoroughly, and then the Optical Density (OD) was measured in a spectrophotometer at wavelengths of (260 nm and 280 nm). The DNA concentration in the solution was calculated according to the following formula: DNA concentration (µg/µl) = [OD260 X 100 (dilution factor) X50 µg/ml] /1000.

Theoretically, OD260 of one corresponds to approximately (50µg/ml) for double strand DNA. The ratio between the reading at 260 nm and 280nm (OD260/OD280) provides an estimation of the purity of nucleic acid (Sambrook et al., 2004). Pure DNA will give an A260/A280 of 1.8 or higher. Values of A260/A280 of less than 1.8 indicate contamination of the DNA by protein. For good PCR results, DNA is required with an A260/A280 quotient of 1.6 or greater (Sambrook et al., 2004; Aumran, 2000; Al-Shummari, 2004; Al-Ouqaili, 2007). 51

Chapter two

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2.4 Amplification Table 2.5 the temperature profile on the instrument was created as follow. Real-time PCR program of HPV 6/11 Step

Temperature oC

Time

Repeats

1

95

15 min

1

2

95

5s

5

60

20 s

72

15 s

95

5s

60

30s fluorescent

3

40

signal detection 72

15s

2.5 Human Papilloma Virus high risk screen Real-TM kit (16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58 and 59) (Sacace, Italy) 2.5.1 Principle of assay Kit HPV High Risk Screen Real-TM Quant is based on two major processes: isolation of DNA from specimens and Real Time amplification. PCR-mix-1 tube contains primers directed against regions of HPV A7, A9 groups (HPV types 16, 18, 31, 33, 35, 39, 45, 52, 58, 59); HPV A5 group (HPV type 51), HPV A6 group (HPV type 56) and βglobine gene used as Internal Control. If the swab was not correctly prepared (high quantity of mucous or insufficient quantity of epithelial cells) the Internal Control will not be detected. The kit contains the quantitative standards with known 52

Chapter two

Materials and Methods

concentration of HPV DNA which allows to determinate the viral load. For the calculation of viral load, the relation between the obtained HPV DNA concentration and the quantity of genomic DNA is used which allows to eliminate the possible errors during the sample preparation.

2.5.2 RT-PCR Protocol: 1. The required quantity of tubes (N+3 (standards) +1(Neg. Control) were prepared. 2. Prepare mix for 40 samples: the tube with PCR-mix-2 buffer 20 μl of Taq F DNA Polymerase was added, mixed by pipette. This mixed was stabled for 3 months at 2-8°C. 3. For each sample tube 7.0 μl of PCR-mix-1-FRT was added and 8.0 μl of Mix (PCR-mix-2 buffer and Taq F DNA Polymerase). 4. 10 μl of extracted DNA sample was added to appropriate tube with Reaction Mix. (Re-centrifuged all the tubes with extracted DNA for 1 min at maximum speed (12000-16000 g) and take carefully supernatant. N.B. don’t disturb the pellet, sorbent inhibit reaction!). 5. For each run 3 standards and 1 Neg Control were prepared: -10 μl of DNA-buffer was added for Negative PCR control; - 10 μl of QS HPV K1was added for tube labeled K1; - 10 μl of QS HPV K2 was added for tube labeled K2; - 10 μl of QS HPV K3 was added for tube labeled K3. 6.

The tubes were closed and transferred into the Real Time PCR

instrument. 7. The samples were positioned and the concentrations of (was reported in the Quant Data Card) in the Joe (Yellow)/HEX, FAM (Green), Rox 53

Chapter two

Materials and Methods

(Orange) and Cy5 (Red) channels in order to generate Standard curves. Using name “Unknown” for the wells that contain samples, K1, K2, K3 for “Standards” and “-” for Negative Controls.

2.5.3 Amplification programme Table 2.6: the temperature profile on the instrument was created as follow

Real-time PCR program of High risk HPV Step

Temperature oC

Time

Repeats

1

95

15 min

1

2

95

5s

5

60

20 s

72

15 s

95

5s

60

30s fluorescent

3

40

signal detection 72

15s

2.5.4 Instrument Settings 2.5.4.1 Plate-type instruments The threshold line should cross only sigmoid curves of signal accumulation of positive samples and should not cross the baseline; otherwise, the threshold level should be raised. Setting the threshold at a level where fluorescence curves are linear and do not cross the curves of the negative samples.

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2.5.4.2 Data Analysis: 1. The experiment may be considered valid if: * The Negative Controls haven’t any positive fluorescence signal; * The standards have positive signals in all channels (Fam, Joe/Hex, Rox, Cy5) 2. The result of the sample is considered: · Invalid in case of absence of any fluorescence signal (positive or internal); · Negative: if signal is present only in the FAM (Green) channel with the concentration of genomic DNA > 5 x 103; · Positive: * For HPV А9 group (16, 31, 33, 35, 52, 58) if contains the positive signal in the Joe (Yellow)/Hex channel with Ct ≤ 33;  for HPV А7 group (18, 39, 45, 59) if it contains the positive signal in the Rox (Orange) channel with Ct ≤ 33;  The concentration was calculated of HPV DNA using the following formula:

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2.5.4.3 Results and Interpretation Table 2.7 the results were interpreted according to the following criteria Result lg (HPV in 100.000 cells)

Interpretation

5

Clinically very important. High risk of cervical dysplasia

*The results can be calculated automatically using the program in Microsoft ® Excel format supplied with the kit.

2.6 Protocol to extract DNA from Fixed formalin paraffin embedded blocks (FFPE) 2.6.1 Procedure DNA was extracted from formalin fixed paraffin embedded tissue as follows:Day 1: 1. One ml of xylol was added (pre incubated at 37 ᵒC for 1hr) incubated at RT for 30min.Then, overtaxing for 30 sec. 2. Spin down for 5min, 13000rpm and supernatant was discarded. 3. The steps 1, 2 &3, were repeated. 4- The pellet was resuspended in 180 μl Buffer ATL. 20 μl proteinase K added (stock solution 20 mg/ml), and mixed by vortexing. 5. Incubation overnight at 56°C in water bath was achieved (until the sample has been completely lysed).

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DAY 2: 6. 200 μl Buffer AL added to the sample and mixed thoroughly by vortexing for 15 sec. 7. If RNA-free genomic DNA was required, three μl RNase A added (100 mg/ml) and incubated for 30 min at room temperature. 8. 200 μl ethanol cooled (96–100%) was added, and mixed again thoroughly by vortexing for 15 sec. and incubated for 5 min at 37ᵒ C. 9. The 1.5 ml tube was briefly centrifuged to removed drops from the inside of the lid. 10. The entire lysate to the QIAamp MinElute column (in a 2 ml collection tube) was carefully transferred without wetting the rim, closing the lid, and centrifuged at 6000 x g (8000 rpm) for 1 min. The QIAamp MinElute column was placed in a clean 2 ml collection tube, and discarding the collection tube containing the flow-through. In some case, the lysate has not completely passed through the membrane after centrifugated, centrifuged again at a higher speed until the QIAamp MinElute column was emptied. 11. The QIAamp MinElute column was carefully opened and 500 μl Buffer AW1 added without wetting the rim. The lid was closed and centrifuged at 6000 x g (8000 rpm) for 1 min. The QIAamp MinElute column was placed in a clean 2 ml collection tube, and discarded the collection tube containing the flow-through. 12. The QIAamp MinElute column was carefully opened and 500 μl Buffer AW2 was added without wetting the rim. The lid was closed and centrifuged at 6000 x g (8000 rpm) for 1 min. The QIAamp MinElute column was placed in a clean 2 ml collection tube, and discarded the collection tube containing the flow-through. The QIAamp MinElute column and the flow-through contact have been avoided.

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13. Carefully open the QIAamp MinElute column and adding 700 μl ethanol (96 -100%) without wetting the rim. Closing the lid and centrifuged at 6000 x g (8000 rpm) for 1 min. Place the QIAamp MinElute column in a clean 2 ml collection tube, and discard the collection tube containing the flow-through. The contact between the QIAamp MinElute column and the flow-through should be avoided. 14. At full speed (20,000 x g; 14,000 rpm) centrifugtion occured for 3 min to dry the membrane completely. This step was necessary, since ethanol carryover into the eluate which may interfere with some downstream applications. 15. The QIAamp MinElute column was Placed in a clean 1.5 ml microcentrifuged tube (not provided), and discarded the collection tube containing the flow-through. The lid of the QIAamp MinElute column was carefully opened and incubated at R.T. for 10 min or at 56 ᵒC for 3min. 16. 30 μl from deionized distilled water was added at room temperature for 5 min, centrifuged at full speed (20,000 x g; 14,000 rpm) for 3 min. 17. The step 17 was repeated and 30 μl from deionized distilled water was added at room temperature for 5 min, at full speed (20,000 x g; 14,000 rpm) was centrifuged for 3 min. After that, incubated at -20 until the samples were used (Sambrook et al., 2004).

2.7 Agarose gel electrophoresis for extracted DNA from Fixed formalin paraffin embedded tissues (FFPE). The DNA extracted products from fixed formalin paraffin embedded tissues (FFPE) and PCR products were detected by agarose gel electrophoresis followed by the detection of the specific bands in ultra violet light.

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Note: Lambda (λ DNA markers): consist of 11 DNA fragments with sizes of 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000 and 1500 bp. It also contains bromophenol blue loading dye. The recommended loading volume was 6 µl/ lane.

2.7.1 Preparation: 1. Ten ml of TBE10x (0.445M Tris –Borate, 0.0125 M EDTA) added to 90 ml of sterile distilled water 2. Two gm agarose gel dissolved in 100ml TBE 1x and prepared by boiling until the solution becomes clear. The solution was allowed to cool to below 60 Cº. Then poured into toped plate to make gel. 3. After polymerization (about 30 minutes at room temperature), the comb and tape were removed. The gel was placed into the gel chamber that was filled with 1x TBE buffer. The gel pocket should be completely covered with buffer. 4. Entire DNA extraction (10μl) was pipetted into the gel wells. 5. Electrophoresis was then carried out for about 1 hour with the following conditions (5 volt/ cm, 100 w and 60 mA). 6. When the electrophoresis was completed; the gel was placed in a tank of staining containing Ethidium bromide (10 mg/ml). Then 0.5μg/ml concentration was prepared according to equation C1V1= C2V2 7. After that the gel was placed on a UV transilluminator (suitable face protection against UV radiation should be worn) 8. A digital picture was made for evaluation and documentation of the results. (Sambrook et al., 2004; Al-Ouqaili, 2007)

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2.7.2 Agarose gel electrophoresis for extracted DNA from Fixed formalin paraffin embedded tissues (FFPE) sample after the amplification DNA extractions from FFPE samples were amplified with real time PCR for high risk HPV genotypes, the PCR products were detected by agarose gel electrophoresis followed by the detection of the specific bands in ultra violet light.

2.7.3 RT-PCR Reaction 2.7.3.1 Reagents were used for PCR reaction: PCR Pre Mix: it contains DNA polymerase, d NTPs, a tracking dye and reaction buffer in premixed format, freeze-dried into pellet. It is ready to use. Note: Before each run, PCR for a control human gene (p53) was done for samples to check the quality of extracted DNA (James et al., 1999).

2.8 Statistical analysis: The data of the 100 cases in this study were entered into and analyzed by the statistical package for social science (SPSS) software version 17.Descriptive statistics were presented as mean, standard deviation for age, purity and DNA concentration. Frequencies and percentages for the other categorical variables. The difference and distribution in age according to the methods of contraception, diagnosis and HPV results were tested, using student t test. The relation of the HPV with education level, contraception and site was tested by using the Chi square test (X2). A level of significance (P value) of ≤ 0.05 was considered significant. (Simon, 2006). 60