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Amatori et al. RT-qPCR array for gene expression

experience, many of these RT-qPCR arrays are induced in HeLa cells, when subjected to CDDP
characterized by technical limits that could compromise or NaBu exposure, was compared with the results
the success of a gene expression study. The first limit obtained by our RT-qPCR array using the same
is represented by the absence of technical replicates biological model of study.
that could monitor the inter-well reproducibility for each
gene transcript. The amplification of each target in METHODS
a single well does not allow one to identify possible
incidental mistakes that can occur, for example, during Cell culture and treatments
preparation of the PCR reaction mix. Another critical HeLa cells were obtained from the American Type
aspect is amplification of genes expressed at very low Culture Collection (ATCC, Rochville, MD, USA).
levels (e.g. CDK inhibitors) that is often difficult and All cellular populations were cultured in Dulbecco’s
requires a highly efficient DNA polymerase. In fact, the Modified Eagle Medium (DMEM, Cambrex,
different DNA polymerase master mixes commercially Walkersville, MD, USA) supplemented with 10% fetal
available are not always able to amplify very low bovine serum (FBS), 1% penicillin-streptomycin, and
represented mRNA species in a detectable way. 1% glutamine.

The rationale of the study was to design and test a RT- HeLa cells in exponential phase of growth were
qPCR array able to analyze the behaviour of 41 human subjected to treatment with 10 µmol/L CDDP (Sigma-
genes involved in cell cycle regulation, DNA damage Aldrich, St Louis, MO, USA) or 5 mmol/L NaBu for
response, apoptosis, and senescence induction. 24 h and 16 h, respectively. At the end of the treatment
Our goal was to develop a qPCR array that could period, viability of cellular populations was analyzed by
overcome the technical limits described above (e.g. the trypan blue dye exclusion method. [11]
low efficiency of amplification and technical replicates
controls) in order to obtain an inexpensive and easy- Primers design
to-use tool for the reliable monitoring of transcriptional Primer pairs, purchased from Primm S.r.l. (Milan,
modulations induced by exposure of human cells to Italy) and listed in Table 1, were designed with
drug treatments. Primer Express 2.0 Abi Prism software (PE Applied
Biosystem, Foster City, CA, USA) as previously
We applied this qPCR array to investigate the response described, employing common design parameters
[12]
of HeLa cells to two different anti-proliferative drugs [Table 2]. All amplicons primers, except for those
such as cisplatin [cis-diamminedichloroplatinum(II)- amplifying an intronic region of GAPDH (control for
CDDP] and sodium butyrate (NaBu). CDDP remains possible genomic DNA contamination), were designed
one of the principal chemotherapeutic agents used for encompassing exon-exon boundaries to avoid genomic
cancer treatment. CDDP cytotoxicity is mediated by DNA amplification. The specificity of amplicons and
its ability to form DNA adducts, primarily intra-strand primer pairs was checked in silico using BLAT (UCSC
adducts, which activate a DNA-damage cellular Genome Browser) and BLAST (National Center for
response and subsequent programmed cell death. [3,4] Biotechnology Information) alignment tools.
Cellular exposure to CDDP, for example, is known
to cause up-regulation of cyclin dependent kinase RNA extraction
inhibitors (e.g. p27 and p21) and down-regulation of Total RNA was extracted using the RNeasy Mini Kit
anti-apoptotic protein Bcl2 in cancer models. [5,6] NaBu (Qiagen, Hilden, Germany) and following supplier’s
is a widely known histone deacetylase inhibitor and is instructions. Elution was performed in a solution of
considered the prototype of an epigenetic modulator. 1 volume of PBS and 5 volumes of RNAlater. For
NaBu exerts anticancer effects by inhibiting histone total RNA extraction RNase-free water and RNase-
deacetylase enzymes, thus inducing increased histone free supplies were used. Total RNA concentration
acetylation levels, elaboration of chromatin structure, was measured with Qubit Fluorometer (Invitrogen,
and consequent reactivation of aberrantly silenced Carlsbad, CA, USA) and RNA integrity evaluated
genes. Previous studies showed that several through 1.3% agarose gel electrophoresis, as
[7]
molecular pathways are affected by NaBu treatment. described. [13]
In particular, cyclin dependent protein kinase (CDK)
down-regulation, cell-cycle inhibitors up-regulation, Reverse transcription
[8]
together with modulation of several apoptosis-related One microgram of RNA from each sample was
genes were observed. [9,10] retrotranscribed (RT) using SuperScript II Reverse
Transcriptase (Invitrogen, Carlsbad, CA, USA) and
Finally, the described modulation of gene expression T Personal thermocycler (Biometra), according to
Journal of Cancer Metastasis and Treatment ¦ Volume 3 ¦ May 24, 2017 91