mil Applications in Oncology
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n=37
n=49
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n=27
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-· ·· ··· ·· · t · · · · ··· ···· t ··· ·· ····· · ~·· · ··· · ·····. ······ ................. .
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Fig. 2. Relative CEA mRNA values for peritoneal washes from the Douglas cavity measured by
real-time RT-PCR with LightCycler in 109 patients with gastric cancer according to the depth
of invasion (pT category). CEA mRNA values correlate statistically with the depth of cancer
invasion (P tumors with mucosal to submucosal invasion, pT2 tumors with muscularis propria to subserosal invasion, pT3 tumors with serosal invasion, and pT4 tumors with invasion to adjacent
tissues
Comments
For normalization of CEA mRNA values, we also quantified GAPDH mRNA as an
internal control by the LightCyder instrument using the hybridization probe format. The CEA/GAPDH ratio correlated well with the depth of tumor invasion, like
the non-normalized data, which suggests that there is no need for correction of
CEA mRNA values with reference to GAPDH mRNA. Since the total number of
cancer cells rather than the cancer cell/noncancer cell ratio in peritoneal washes
may be the hallmark of progression of metastatic spread, we used uncorrected
CEA mRNA rather than the CEA/GAPDH ratio for a more convenient and appropriate parameter to monitor peritoneal recurrence in this study.
The key to successful quantification, especially in the low copy range, lies in
the storage of the external standards. We prefer to prepare multiple aliquots, in
order to avoid repetitive freezing and thawing. We dilute them to our working
concentration and store them in aliquots at -30°C. For long-term storage, -BO°C
is optimal.