Esophageal Adenocarcinoma Methods and Protocols

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  1. Droplet generator (e.g., QX200 Droplet Generator).

  2. Droplet reader (e.g., QX200 Droplet Reader).

  3. Microfluidic cartridge.

  4. Gaskets.

  5. Generation oil (e.g., Bio-Rad Droplet Generation Oil for
    Probe).

  6. Microcentrifuge machine.

  7. Computer.

  8. QuantaSoft software.


3 Methods



  1. Esophageal adenocarcinoma tissues are cryotomed into five
    slices of 5 μM sections.

  2. Place a section onto a microscope slide for hematoxylin and
    eosin (H&E) staining to ensure pathological confirmation (see
    Note 1).

  3. Extract genomic DNA from an esophageal tissue sample using
    commercially available tissue extraction kit according to the
    manufacturer’s guideline (see Notes 2 and 3 ).

  4. Measure A 260 /A 280 ratio and A 260 /A 230 ratio of the resulting
    elution using a nanospectrophotometer (see Note 4).

  5. Ensure that the A 260 /A 280 ratio is approximately 1.8 and
    A 260 /A 230 ratio is between 2.0 and 2.2 (see Note 5).

  6. Store extracted DNA at − 20 °C (see Note 6).

  7. Both primer sets for the target gene and control gene are
    designed to be approximately 80 and 60 bp individually.
    Primers can be designed using a variety of available tools rang-
    ing from paid software to freeware such as the NCBI (National
    Centre for Biotechnology Information) primer design tool
    (https://www.ncbi.nlm.nih.gov/tools/primer-blast/) which
    uses Primer3 and BLAST to help design the primers (Fig. 1 ).

  8. Briefly, enter the accession, gi or FASTA sequence of your gene
    of interest at the PCR template section (Fig. 1 ).

  9. Next, correct the PCR product size to 60 or 80 bp at the
    Primer Parameters section (Fig. 1 ).

  10. Click on Advanced parameters at the bottom of Get Primers
    button (Fig. 1 ) and scroll to internal hybridization oligo
    parameters section (Fig. 2 ) and tick on the Pick internal hybrid-
    ization oligo to design your probes.

  11. Fill up any other additional criteria of your preference and then
    hit Get Primers (Fig. 2 ).


3.1 Genomic DNA
Extraction


3.2 Droplet Digital
PCR (ddPCR) Primer
Design


Katherine T. W. Lee et al.
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