Method Article

Simple and Rapid Method to Obtain High-quality Tumor DNA from Clinical-pathological Specimens Using Touch Imprint Cytology

DOI:

10.3791/56943

⸱

March 21st, 2018

 ,  ,  , 
1Genome Analysis Center, Yamanashi Central Hospital, 2Pathology Division, Laboratory Department, Yamanashi Central Hospital, 3The University of Tokyo

In This Article

Summary

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Obtaining high-quality genomic DNA from tumor tissues is an essential first step for analyzing genetic alterations using next generation sequencing. In this article, we present a simple and rapid method to enrich tumor cells and obtain intact DNA from touch imprint cytology specimens.

Abstract

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It is critical to determine the mutational status in cancer before administration and treatment of specific molecular targeted drugs for cancer patients. In the clinical setting, formalin-fixed paraffin-embedded (FFPE) tissues are widely used for genetic testing. However, FFPE DNA is generally damaged and fragmented during the fixation process with formalin. Therefore, FFPE DNA is sometimes not adequate for genetic testing because of low quality and quantity of DNA. Here we present a method of touch imprint cytology (TIC) to obtain genomic DNA from cancer cells, which can be observed under a microscope. Cell morphology and cancer cell numbers can be evaluated using TIC specimens. Furthermore, the extraction of genomic DNA from TIC samples can be completed within two days. The total amount and quality of TIC DNA obtained using this method was higher than that of FFPE DNA. This rapid and simple method allows researchers to obtain high-quality DNA for genetic testing (e.g., next generation sequencing analysis, digital PCR, and quantitative real time PCR) and to shorten the turnaround time for reporting results.

Introduction

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Next generation sequencing technology has provided researchers significant advancements in analyzing genome information in genetic variations, Mendelian disease, hereditary predisposition, and cancer 1,2,3. The Cancer Genome Atlas (TCGA) and International Cancer Genome Consortium (ICGC) have pursued the identification of genetic alterations in several types of common cancers4. Hundreds of essential cancer driver genes have been successfully identified, and some of these molecules are being targeted for drug development1,5,6.

In the clinical setting, FFPE specimens are commonly used for pathological diagnosis and molecular testing for various diseases, including cancer. However, during the fixation process with formalin, DNA-protein or DNA-DNA cross-linking occurs and DNA fragmentation is induced. Thus, FFPE DNA samples are not always suitable for genetic analysis because of low quality and quantity of DNA7,8,9. Additionally, it takes several days to prepare FFPE specimens, and technical skill is necessary to accurately prepare the sections. Therefore, it is desirable to develop a simple and rapid method for obtaining high-quality intact DNA.

Cytology is an alternative method for pathological diagnosis. Cytological sample preparation is a simpler, less expensive, and more rapid approach compared with FFPE preparation10. The TIC technique has been performed on sentinel lymph nodes and marginal tissues from breast cancer patients for intraoperative rapid diagnosis for some years11,12. However, there are few reports that have examined whether high-quality genomic DNA can be extracted from TIC specimens and used for subsequent genetic analysis. Cytological specimens are commonly stained with Papanicolaou (Pap) or Giemsa staining, and we previously reported that the amount and quality of DNA extracted from TIC specimens (especially Giemsa-stained samples) are superior to samples obtained from FFPE tissues13. Compared with Pap staining, Giemsa staining has an advantage in requiring less staining procedures. In Pap staining, after the samples have been fixed and stained, they must be mounted with mounting medium (e.g., Malinol) for distinguishing sample contents, such as tumor cells, normal cells, and inflammatory cells under a microscope. If the Pap specimen is prepared without the mounting step, it is almost impossible to observe the cells under a microscope because the specimen is dried. In comparison, Giemsa staining can be observed in the dried state, therefore, the mounting step is not necessary for quick cellular evaluation. For microdissection, Giemsa staining is more suitable because it requires dry specimens.

In this report, we introduce a simple and rapid method for preparing TIC specimens with Giemsa staining and demonstrate that TIC is a better source for DNA compared with FFPE specimens.

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Protocol

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1. TIC Preparation for Quick Microscopic Assessment Using Normal Glass Slides

  1. Perform the TIC preparation as soon as possible after clinical pathological tissue materials are available. If TIC specimens cannot immediately be prepared, keep the tissue materials covered with saline moistened sterile-gauze and store in the refrigerator to prevent drying of tissues.
  2. Prepare 5 mm3 tissue material such as solid tumors (e.g., liver, lung, and breast tissues) clinically obtained by surgery or endoscopy.
    1. Gently wipe the tissue with sterile-gauze coated with physiological saline and remove blood, if the tissue surface has a lot of blood.
    2. For microscopic specimens such as biopsy material, keep the sample moistened with sterile-gauze soaked in physiological saline.
  3. Cut and trim the normal tissue with a trimming knife and expose the surface of the tumor lesion, if the tumor masses are not visible grossly.
  4. Touch the tumor surface of the resected specimens onto a normal glass slide several times with gloved hands. Visually confirm the touched area is over 80% of the normal glass slide.
  5. Lightly press the normal glass slide against a polyethylene naphthalate (PEN) membrane slide and rub gently 2 - 3 times with gloved hands. Visually confirm the cells are transferred from the normal glass slide to the PEN membrane slide.
  6. Air-dry both the glass and PEN membrane slides for 5 min at room temperature.
  7. Stain the normal glass slide for direct cytological examination. Dip the glass slides with fixative solution for 5 s, and then stain with Giemsa staining solution for 15 s.
  8. Assess and screen the tumor contents and cellularity on the normal glass slide entirely with a microscope for quick assessment. Evaluate tumor cells based on several criteria; nuclear enlargement, abnormal karyotype, abundant chromatin, unequal distribution of cells, ratio of nuclear component/cytoplasmic component, cell size, and cell polarity.

2. Preparation of the PEN Membrane Slide Film for Genetic Testing

  1. If the sample shows tumor cellularity over 60% by quick microscopic assessment (step 1.8), cut the tumor-touched film of the PEN membrane slides for DNA extraction with a knife and gloved hands. Transfer the cut film to a sterile microcentrifuge tube with a pincette and gloved hands.
  2. If the tumor cellularity was determined as low (less than 60% of the tumor contents) by quick microscopic assessment (step 1.8), use laser capture microdissection and obtain tumor samples.
    1. Perform Giemsa staining to assess the tumor cells using standard protocols.
    2. Cut the film of the PEM membrane slide by appropriate laser capture microdissection.
    3. Transfer the cut film to a sterile microcentrifuge tube with a pincette and gloved hands.
    4. Store the film-containing microcentrifuge tube at 4 °C until DNA extraction (the protocol can be paused here).

3. DNA Extraction

  1. Perform DNA extraction from the TIC or FFPE tissue samples using a FFPE DNA extraction kit according to the manufacturer's instructions with minor modifications. An equivalent kit is available for the FFPE DNA extraction step.
  2. Add 180 µL of tissue lysis buffer (pH = 8.3) to the film-containing microcentrifuge tube with a manual 200-µL pipette. Add 20 µL of proteinase K with a manual 20-µL pipette and mix by vortexing with a vortex mixer at maximum speed (approximately 2,500 rpm) for 5 s.
  3. Incubate the samples at 56 °C overnight with an air incubator.
  4. Incubate the FFPE and TIC samples at 90 °C in a heat block for 1 h and 10 min, respectively. Briefly spin down the microcentrifuge tube at 1,500 x g for 5 s at room temperature with a mini centrifuge.
  5. Add 200 µL of lysis buffer to the sample with a 200-µL pipette and mix thoroughly by vortexing at maximum speed for 5 s.
  6. Add 200 µL of ethanol (96 - 100%) with a 200-µL pipette and mix thoroughly by vortexing at maximum speed for 5 s. Briefly spin down the microcentrifuge tube at 1,500 x g for 5 s at room temperature with a mini centrifuge.
  7. Carefully transfer the entire lysate to the spin column with a 1,000-µL pipette and centrifuge at 6,000 x g for 1 min at 25 °C.
  8. Place the spin column in a clean 2-mL collection tube with gloved hands, and discard the collection tube containing the flow-through into a plastic disposal box.
  9. Add 500 µL of wash buffer to the spin column with a 1,000-µL pipette and centrifuge at 6,000 x g for 1 min at 25 °C.
  10. Place the spin column in a clean 2-mL collection tube with gloved hands, and discard the collection tube containing the flow-through into a plastic disposal box.
  11. Add 500 µL of wash buffer to the spin column with a 1,000-µL pipette and centrifuge at 6,000 x g for 1 min at 25 °C.
  12. Discard the collection tube containing the flow-through into a plastic disposal box. Place the spin column in a clean 1.5-mL microcentrifuge tube with gloved hands and centrifuge at 20,000 x g for 3 min at 25 °C to dry the membrane.
  13. Place the spin column in a DNA-low binding tube with gloved hands.
    1. Add 40 - 50 µL of elution buffer to the center of the membrane with a 100-µL pipette.
    2. Incubate at room temperature for 5 min and centrifuge at 20,000 x g for 1 min at 25 °C.
    3. Store the DNA samples at -20 °C until the next step (the protocol can be paused here).

4. Estimation of DNA Quality by Quantitative Real Time PCR

  1. Prepare the master mix in a sterile microcentrifuge tube with a pipette, as follows: 10 µL of 2x Real-Time PCR Master Mix, 1 µL of 20x RNase P Primer-Probe Mix (amplicon size: 87 bp), and 8 µL of sterile nuclease-free water.
  2. Prepare the second master mix in one sterile microcentrifuge tube as follows: 10 µL of 2x Real-Time PCR Master Mix, 1 µL of 20x RNase P Primer-Probe Mix (amplicon size: 268 bp), and 8 µL of sterile nuclease-free water.
  3. Perform serial dilutions of human control genomic DNA (supplied in the kit) 4 times for a five-point standard curve and determine the absolute DNA concentrations13.
  4. Add 19 µL of the two prepared master mixes (prepared in step 4.1 and 4.2) into separate wells of an optical 96-well reaction plate with a 20-µL pipette.
  5. Add 1 µL of FFPE DNA or TIC DNA to separate wells containing the reaction mix with a 2-µL pipette. Add 1 µL of nuclease-free water into a separate well containing the reaction mix for the no template control.
  6. Hold the non-adhesive side of an optical adhesive film and peel back the protective backing from the center of the film. Gently drag the applicator over the film and seal the film over the 96-well plate.
  7. Gently mix the 96-well plate using a 96-well plate mixer for 10 s at room temperature at 2,000 rpm. Centrifuge the plate briefly at 1,000 x g for 3 min at room temperature.
  8. Power on the real-time PCR instrument and insert the 96-well plate. Run the PCR reactions using the following protocol: 95 °C for 20 s, followed by 45 cycles of 95 °C for 1 s and 60 °C for 20 s. Use "standard curve" and "fast mode."
  9. Assess DNA fragmentation with the ratio of DNA (relative quantification; RQ) obtained for the long amplicon (268 bp) to the short amplicon (87 bp). RQ is the mean value of the long amplicon/the mean value of the short amplicon13.

5. Preparation of the Next Generation Sequencing Library

  1. Prepare the sequencing library for next generation sequencing according to the manufacturer's instructions.
  2. Prepare the multiplex PCR master mix in a sterile microcentrifuge tube per sample as follows: 4 µL of 5x Multiplex PCR reaction solution, 4 µL of 5x primer pool, ≤6 µL TIC or FFPE DNA (1 - 100 ng), and add nuclease-free water up to 20 µL.
    1. Add the multiplex PCR master mix to a PCR tube and mix gently by tapping the tube.
    2. Briefly spin down the microcentrifuge tube at 1,500 x g for 5 s at room temperature with a mini centrifuge.
  3. Run the PCR reactions using the following protocol: 99 °C for 2 min, followed by 20 cycles of 99 °C for 15 s and 60 °C for 4 min, and holding step 10 °C. Briefly spin down the PCR tube with a mini centrifuge at 1,500 x g for 5 s at room temperature.
    NOTE: Determine the number of cycles based on the number of primer pairs.
  4. Open the lid of the PCR tube, and add 2 µL of the restriction enzyme with a 2-µL pipette. Close the lid of PCR tube and mix gently by tapping the PCR tube. Briefly spin down the PCR tube with a mini centrifuge at 1,500 x g for 5 s at room temperature.
  5. Run the PCR reactions using the following protocol: 50 °C for 10 min, 55 °C for 10 min, 60 °C for 20 min, and holding step 10 °C. Briefly spin down the PCR tube with a mini centrifuge at 1,500 x g for 5 s at room temperature.
  6. Add the adaptor ligation master mix into the each well containing the digested PCR amplicons with a pipette as follows: 4 µL of adaptor ligation solution, 0.5 µL of barcode, 0.5 µL of adaptor, 2 µL of nuclease-free water, and 2 µL of DNA ligase. Close the lid of PCR tube and mix gently by tapping. Briefly spin down the PCR tube with a mini centrifuge at 1,500 x g for 5 s at room temperature.
  7. Run the PCR reactions using the following protocol: 22 °C for 30 min, 68 °C for 5 min, 72 °C for 5 min, and holding step 10 °C.
  8. Purify the sequencing library with magnetic beads according to the manufacturer's instructions.
  9. Transfer the adaptor-ligated library solution into the 1.5-mL DNA low-binding tube. Add 45 µL of magnetic beads into the DNA low-binding tube for the 1st purification. Mix gently by tapping the tube and incubate for 5 min at room temperature.
  10. Place the DNA-low binding tube in a magnetic rack, then incubate for 2 min at room temperature until the solution is clear. Carefully discard the supernatant with a 200-µL pipette without disturbing the magnetic beads.
  11. Add 150 µL of freshly prepared 70% ethanol with a 200-µL pipette, then move the tube side-to-side of the magnet to wash the beads. Carefully discard the supernatant without disturbing the magnetic beads.
  12. Repeat step 5.11 for a second wash.
  13. Briefly spin down the tube with mini centrifuge at 1,500 x g for 5 s at room temperature. Place the DNA low-binding tube in a magnetic rack, and carefully discard the ethanol droplets with a 10-µL pipette.
  14. Add 50 µL of Low TE into the DNA low-binding tube containing the magnetic beads pellet to disperse the beads. Incubate for 2 min at room temperature.
  15. Place the DNA low-binding tube in a magnetic rack, and incubate at room temperature for 2 min until the solution is clear.
  16. Transfer the 50 µL of supernatant into the new DNA low-binding tube and add 75 µL of magnetic beads with a 100-µL pipette for the 2nd purification. Mix gently by tapping the tube and incubate for 5 min at room temperature.
  17. Place the DNA low-binding tube in a magnetic rack, then incubate for 2 min at room temperature until the solution is clear. Carefully discard the supernatant with a 200-µL pipette without disturbing the magnetic beads.
  18. Add 150 µL of freshly prepared 70% ethanol with a 200-µL pipette, then move the tube side-to-side of the magnet to wash the beads. Carefully discard the supernatant without disturbing the magnetic beads.
  19. Repeat step 5.18 for a second wash.
  20. Briefly spin down the tube with a mini centrifuge at 1,500 x g for 5 s at room temperature. Place the DNA low-binding tube in a magnetic rack, and carefully discard the ethanol droplets with a 10-µL pipette.
  21. Add 50 µL of low TE into the DNA low-binding tube containing the magnetic beads pellet to disperse the beads. Incubate for 2 min at room temperature.
  22. Place the DNA low-binding tube in a magnetic rack, and incubate at room temperature for 2 min until the solution is clear.
  23. Transfer the 45 µL of supernatant containing purified library into the new DNA low-binding tube with a 100-µL pipette.

6. Quantify the Library Concentration by Quantitative Real Time PCR

  1. Determine the concentration of each library according to the manufacturer's instructions13.
    1. Prepare a 20-fold dilution solution as follows: mix 2 µL of purified library and 38 µL of nuclease-free water in a DNA low-binding tube with a 2-µL and 100-µL pipette.
    2. Store the undiluted libraries at -20 °C until step 7.3.
  2. Prepare a 200-fold dilution solution as follows: mix 5 µL of the 20-fold diluted purified library (prepared in step 6.1) and 45 µL of nuclease-free water in a DNA low-binding tube.
  3. Prepare a 2,000-fold dilution solution as follows: mix 5 µL of the 200-fold diluted purified library (prepared in step 6.2) and 45 µL of nuclease-free water in a DNA low-binding tube.
  4. Prepare the reaction master mix as follows: mix 10 µL of 2x master mix solution and 1 µL of 20x primer-probe assay solution in sterile microcentrifuge tube with a pipette, then mix by tapping the tube. Add 11 µL of the reaction master mix into the wells of an optical 96-well reaction.
  5. Add 9 µL of the 2,000-fold diluted library, 9 µL of each standard control, or 9 µL of nuclease-free water to each well with a 10-µL pipette.
  6. Hold the non-adhesive side of optical adhesive film and peel back the protective backing from the center of the film.
    1. Gently drag the applicator over the film and seal the film over the 96-well plate.
    2. Gently mix the 96-well plate using a 96-well plate mixer for 10 s at room temperature.
    3. Centrifuge the plate briefly at 1,000 x g for 3 min at room temperature.
  7. Power on the real-time PCR instrument and insert the 96-well plate. Run the PCR reactions using the following protocol: 50 °C for 2 min, 95 °C for 20 s, followed by 40 cycles of 95 °C for 1 s and 60 °C for 20 s. Use "standard curve" and "fast mode."
  8. Calculate the undiluted library concentration by multiplying the concentration determined with qPCR by 2,000.

7. Next Generation Sequencing

  1. Plan the run condition and set the run parameter within the software.
    1. Click [Plan tab] and [Template], and select the appropriate run method.
    2. Select the application and technique type, and click [Next].
    3. Select the instrument, sample preparation kit (optional), library kit type, template kit, sequencing kit, base calibration mode, chip type, control sequence (optional), and barcode set, and click [Next].
    4. Select plugins and click [Next].
    5. Select project and click [Next].
    6. Select default reference and BED files of the targeted region.
    7. Type the sample name, select the barcode, and click [Plan Run].
  2. Perform template preparation and chip loading in an automated instrument according to the manufacturer's instructions. Thaw the reagent cartridge at room temperature for 45 min before use.
  3. Dilute the undiluted library with nuclease-free water according to the library concentration calculated in step 6.8 and make 20 pM libraries.
    1. Prepare a pooled library for sequencing and store on ice.
    2. Add 25 µL of the pooled library with a 100 µL pipette to the bottom of the sample tube. Use the pooled library within 48 h.
  4. Power on and open the cover of the automated instrument.
    1. Place the sequencing chip, chip adaptor, enrichment cartridge, tip cartridge, PCR plate, PCR frame seal, recovery tube, solution cartridge, and reagent cartridge to the appropriate position of the automated instrument.
    2. Touch the [Set up Run] and [Step by step] on the screen.
    3. Close the cover and touch [Start check] on the screen.
    4. After the deck scan process, touch [Next] on the screen.
    5. Check the display contents (kit type, chip type, chip ID, sample ID, plans), set the time, and touch [OK] on the screen.
  5. After finishing the chip loading:
    1. Touch [Next] on the screen and open the cover.
    2. Unloaded the sequencing chip from chip adaptor with gloved hands.
    3. Place the chip into the chip container, rap with parafilm and store at 4 °C until the sequencing reaction.
    4. Removed the enrichment cartridge, PCR plate, PCR frame seal, recovery tube, solution cartridge, and reagent cartridge from the appropriate position of the automated instrument with gloved hands.
    5. Transfer an empty-tip cartridge to the waste tip position of the automated instrument with gloved hands.
    6. Touch [Next] and close the cover.
    7. Touch [Start] and clean the automated instrument by ultraviolet rays for 4 min.
  6. Dissolve a sodium chlorite tablet in 1,000 mL of ultrapure water and filter solution with a 0.22-µm filter flow filter unit.
    1. Power on the sequencing instrument.
    2. Touch [Clean] and [Next] on the screen of the sequencing instrument.
    3. Clean the sequencing instrument with 250 mL of filter-sterilized sodium chlorite solution and subsequently 250 mL of ultrapure water.
  7. Touch [Initialize] and select the appropriate sequencing kit on the screen.
    1. Install a gray shipper at the appropriate location with gloved hands.
    2. Initialize the sequencing instrument with the wash solution (provided in the kit), the pH adjustment solution (containing 350 µL of 100 mM sodium hydroxide), and the pH standard solution (provided in the kit).
  8. Add 20 µL of dATP, dGTP, dCTP, and dTTP nucleotides (provided in the kit) in 50-mL tubes (provided in kit) with a 100-µL pipette.
    1. Install a gray shipper at the appropriate location with gloved hands.
    2. Load the 50-mL tube and screw onto the sequencing instrument.
    3. Touch the [Next] on the screen to start the initialization step, which takes approximately 25 min.
  9. After completing the initialization step:
    1. Touch [Run] on the screen and select the appropriate library preparation instrument.
    2. Scan the two-dimensional barcode of the chip.
    3. Insert the sequencing chip on the appropriate position.
    4. Close the chip clamp and instrument door.
    5. Touch [Chip check], [Next], and [OK] on the screen to start the sequencing run.
  10. After the sequencing reaction, transfer the data and perform data-analyzing pipeline on the sequencing server13,17.

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Results

$$\rightleftharpoonup{xx}$$ $$\longleftharp{xx}$$, $$\longrightharp{xx}$$,

Figure 1 shows the entire process from preparing TIC specimens to DNA extraction. Notably, the procedure takes only two days to obtain genomic DNA from TIC samples. We evaluated any effects of tumor storage before the slide processing. We found that tumor cells were attached onto the glass slide when tissue specimens were immediately touched onto the slide, and when tissues were kept in saline moistened sterile-gauze for 1 h (Figure 2

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Discussion

$$\rightleftharpoonup{xx}$$ $$\longleftharp{xx}$$, $$\longrightharp{xx}$$,

In this study, we presented an alternative method for obtaining tumor DNA from clinical pathological specimens using TIC. TIC preparation is very simple and needs less time compared with FFPE methods, without the requirement for special instruments10. All procedures from the TIC preparation to DNA extraction can be completed within two days (Figure 1). This method thus shortens the turnaround time for performing genetic testing. Notably, this provides a significant ad...

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Disclosures

$$\rightleftharpoonup{xx}$$ $$\longleftharp{xx}$$, $$\longrightharp{xx}$$,

The authors have nothing to disclose.

Acknowledgements

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We thank all the medical and ancillary staff of the hospital and the patients for consenting to participate. We thank Gabrielle White Wolf, PhD, from Edanz Group (www.edanzediting.com/ac) for editing a draft of this report. This study was supported by a Grant-in-Aid for Genome Research Project from the Yamanashi Prefecture (Y.H. and M.O.) and a grant from The YASUDA Medical Foundation (Y.H.).

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Materials

List of materials used in this article
NameCompanyCatalog NumberComments
FINE FROST white20 micro slide glassMatsunami Glass ind, LtdSFF-011
Arcturus PEN Membrane Glass SlidesThermo Fisher ScientificLCM0522
Cyto Quick A solutionMuto Pure Chemicals20571
Cyto Quick B solutionMuto Pure Chemicals20581
May-Grunwald SolutionMuto Pure Chemicals15053
Giemsa solutionMuto Pure Chemicals15002
QIAamp DNA FFPE tissue kitQiagen56404
TaqMan Fast Advanced Master MixThermo Fisher Scientific4444557
TaqMan RNase P Detection Reagents KitThermo Fisher Scientific4316831
TaqMan Assay from FFPE DNA QC Assay v2Thermo Fisher Scientific4324034
MicroAmp Fast Optical 96-Well Reaction Plate Thermo Fisher Scientific4346907
MicroAmp optical Adhesive FilmThermo Fisher Scientific4311971
MicroMixer E36TITEC0027765-000
ViiA 7 Real-Time PCR SystemThermo Fisher ScientificVIIA7-03
Himac CF16RXIIHitachi-kokiCF16RII
Ion Library TaqMan Quantitation KitThermo Fisher Scientific4468802
Ion AmpliSeq Cancer Hotspot Panel v2Thermo Fisher Scientific4475346
Ion AmpliSeq Library Kit 2.0Thermo Fisher Scientific4480442
Ion Xpress Barcode Adapters 1-16 KitThermo Fisher Scientific4471250
Ion PGM Hi-Q View Sequencing Kit (200 base)Thermo Fisher ScientificA30044
Ion Chef SystemThermo Fisher Scientific4484177
Veriti 96-well Thermal CyclerThermo Fisher ScientificVeriti200
Ion 318 Chip Kit v2 BCThermo Fisher Scientific4488150
Ion PGM SystemThermo Fisher ScientificPGM11-001
Ion PGM Wash 2 Bottle kitThermo Fisher ScientificA25591
Agencourt™ AMPure™ XP KitBeckman CoulterA63881
16-position Magnetic StandThermo Fisher Scientific4457858
Nonstick, RNase-free Microfuge Tubes, 1.5 mL (Low binding tube)Thermo Fisher ScientificAM12450
Nuclease-free waterThermo Fisher ScientificAM9938
MicroAmp™ Optical 96-well Reaction PlatesThermo Fisher Scientific4306737
MicroAmp™ Clear Adhesive FilmThermo Fisher Scientific4306311
Agencourt™ AMPure™ XP KitBeckman CoulterA63881
Ethanol(99.5)Nacalai Tesque08948-25
Sodium hydroxide (10M)Sigma72068
DTU-NeoTAITEC0063286-000
E-36TAITEC0027765-000
ECLIPSE Ci-LNikon704354
Pipet-Lite LTS Pipette L-2XLS+METTLER TOLEDO17014393
Pipet-Lite LTS Pipette L-10XLS+METTLER TOLEDO17014388
Pipet-Lite LTS Pipette L-20XLS+METTLER TOLEDO17014392
Pipet-Lite LTS Pipette L-100XLS+METTLER TOLEDO17014384
Pipet-Lite LTS Pipette L-200XLS+METTLER TOLEDO17014391
Pipet-Lite LTS Pipette L-1000XLS+METTLER TOLEDO17014382
petit-changeWAKENMODEL8864Mini centrifuge
petit-incubatorWAKENWKN-2290Air incubator
SensiCare Powder-free Nitrile Exam GlovesMEDLINESEM486802
Sterile gauzeOsaki11138
Refrigerator MediCoolSANYOMPR-312DCN-PJ
FEATHER TRIMMING BLADFEATHERNo.130
FEATHER TRIMMING BLADFEATHERNo.260
FEATHER SFEATHERFA-10
Vortex Genius 3IKA41-0458Vortex mixer
PincetteNATSUMEA-5
1.5 mL microtubeBIOBIKRC-0150

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Tumor DNA ExtractionFFPE DNA ComparisonPEN Membrane SlideDNA Quality AssessmentNext Generation SequencingDigital PCRQuantitative Real Time PCRSomatic Mutation DetectionAutomated Sequencing

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