200
HER-2 has a higher survival rate when treated with anti-HER-2
targeted therapy (trastuzumab) [ 48 , 49 ]. Therefore, further iden-
tifying the altered genomic target can help clinician in choosing
the best treatment method.
On the other hand, Li and colleagues found that the somatic
genomes of patients with Barrett’s esophagus that did not progress
to esophageal adenocarcinoma were exceptionally stable through-
out the observation period. However, genomic alterations were
progressively observed in patients with Barrett’s esophagus up to
48 months before esophageal adenocarcinoma development, par-
ticularly on chromosome 18q [ 50 ].
Overall, the values of detecting the copy-number change high-
light the importance of somatic copy-number alterations analysis.
In the past, copy-number alterations have been mostly analyzed
with the use of real-time PCR (qPCR). However, with recent
advances in technology, several new methods such as next genera-
tion sequencing and digital PCR (dPCR) could identify accurately
the somatic copy-number alterations. In this chapter, we will be
focusing on the use of dPCR to quantify accurately somatic copy-
number alteration in patients with esophageal adenocarcinoma. It
is more sensitive than standard PCR method or next generation
sequencing in detecting the DNA copy number.
While the concept of this technology came from two decades
ago [ 51 , 52 ], this method allows for absolute quantification of
targets contained in a sample without the prerequisite of huge
technical replicates. dPCR workflow is relatively similar to that of
quantitative real-time PCR (qPCR) wherein the reaction mixture
containing sample nucleic acid, primers and/or probes plus the
master-mix are prepared accordingly. Unlike qPCR though, dPCR
first partitions samples into tens of thousands of minute size reac-
tion chamber prior to thermal cycling to the endpoint and count-
ing the positive signals versus the negative signals [ 51 – 54 ].
Currently, there are two ways of partitioning the samples, one
through physical partitioning chambers [ 54 ] and another through
droplet generation via water-oil emulsion concept [ 55 ]. In the fol-
lowing section, we describe the emulsion-based method of dPCR
known as droplet digital PCR (ddPCR).2 Materials
Prepare all reagents using ultrapure water and analytical grade
chemicals. Ensure all plastic wares and reagents are DNase-free.
Unless otherwise indicated, prepare and store all reagents at room
temperature. Follow the rules and regulations for waste disposal.
Appropriate personal protective equipment must be worn during
experiments to safeguard oneself against laboratory dangers.Katherine T. W. Lee et al.