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Amatori et al. RT-qPCR array for gene expression

qPCR array using SYBR-green master mix that showed elaborations have been widely documented in HeLa
the best efficiency in our conditions. This allowed us to cells treated by CDDP. [5,6,17,18] In addition, a significant
monitor transcript levels of low-expressed genes (e.g. down-regulation of anti-apoptotic gene BCL2 also
CDKN2B) [Figure 2B-D]. confirmed previously reported data. [5,19] All these
modulations were largely expected since CDDP is
Then, the RT-qPCR array was validated using known to exert its activity by targeting DNA of cells
HeLa cells subjected to treatment with two different and forming covalent adducts that lead to activation
drugs, CDDP and NaBu, known to induce important of the cellular DNA damage response. In fact, once
[4]
modulations of the gene expression profile of this damaged, cells respond by inhibiting its progression
cellular model. through the cell cycle and, in case of extensive damage,
by activating the apoptotic cell death program.
In particular, CDDP treatment was able to up-regulate
growth arrest and DNA damage response genes Interestingly, we found that CDDP also induces up-
GADD45A and BRCA1, as well as cell cycle inhibitors regulation of cyclin dependent kinases 1 and 2 (CDK1
genes CDKN1A and CDKN2B. These transcriptional and CDK2). This result, which is to our knowledge the

Figure 5: Application of the PCR array to sodium butyrate (NaBu)-treated HeLa cells. Difference in transcriptional activity of sodium
butyrate-treated HeLa cells, compared to non-treated cells, evaluated by 2 -∆∆CT method. Data reported as mean ± standard deviation of
3 independent experiments. (A) Relative expression of differentially expressed genes; (B-E) examples of qPCR amplification plots and
melting curves of differentially expressed genes. PCR: polymerase chain reaction
Journal of Cancer Metastasis and Treatment ¦ Volume 3 ¦ May 24, 2017 97