202
- Droplet generator (e.g., QX200 Droplet Generator).
- Droplet reader (e.g., QX200 Droplet Reader).
- Microfluidic cartridge.
- Gaskets.
- Generation oil (e.g., Bio-Rad Droplet Generation Oil for
Probe). - Microcentrifuge machine.
- Computer.
- QuantaSoft software.
3 Methods
- Esophageal adenocarcinoma tissues are cryotomed into five
slices of 5 μM sections. - Place a section onto a microscope slide for hematoxylin and
eosin (H&E) staining to ensure pathological confirmation (see
Note 1). - 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 ). - Measure A 260 /A 280 ratio and A 260 /A 230 ratio of the resulting
elution using a nanospectrophotometer (see Note 4). - 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). - Store extracted DNA at − 20 °C (see Note 6).
- 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 ). - Briefly, enter the accession, gi or FASTA sequence of your gene
of interest at the PCR template section (Fig. 1 ). - Next, correct the PCR product size to 60 or 80 bp at the
Primer Parameters section (Fig. 1 ). - 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. - 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.