Biology Reference
In-Depth Information
4. Optical adhesive fi lms (Life Technologies).
5. RNase DNase-free water (Life Technologies).
6. Real-Time PCR System 7300 (Life Technologies).
3
Methods
To apply DNA microarray technology for the transcriptome study
in response to adenoviral vector transduction, some issues must be
taken into consideration to optimize the reproducibility, reliability,
and sensitivity of the analysis. First, in order to obtain reproducible
data, cells have to be treated with a vector dose that guarantees
homogeneous and signifi cant transduction. In human cells, in
vitro, we suggest a dosage of 1,000 adenovector genomes per cell
that, in average, ensures at least 50 % of transduction for most of
the cell types [
10
]. A second aspect concerns cell cultures: in order
to avoid background transcriptome variability due to the different
cell donor and/or passage, we suggest to create a cell bank on
which to systematically perform experiments. A further aspect to
examine, is the number of replicates needed to obtain reliable
results. To obtain reliable estimates of modulation between sam-
ples and controls in terms of
p
-value and false discovery rates,
assuming that outliers do not occur, three to fi ve gene chips per
group are usually adequate. If outliers did occur, the sample should
be discarded and repeated. Lastly, another key issue to generate
reliable transcriptome data concerns the isolation and processing
of DNase-free and high-quality RNA.
1. The hepatocyte-derived cellular carcinoma cell line Huh7 is
cultured in complete medium supplemented with penicillin
and streptomycin and incubated at 37 °C, 5 % CO
2
. When
approaching confl uence, Huh7 are passaged with trypsin/
EDTA to provide new maintenance cultures on 100 mm tissue
dishes and experimental cultures on 60 mm tissue dishes.
A 60 mm culture dish is required for each experimental point.
A 1:3 split of Huh7 cells provides experimental cultures that
reach the confl uence after 48-72 h.
2. Human midbrain neuronal progenitor cells (hmNPCs) are
cultured in expansion medium as in ref. [
13
]. hmNPCs are
maintained in 5 % CO
2
, 3 % O
2
and 37 °C in T-25 cm
2
or
T-75 cm
2
fl asks coated 3 h with 15
3.1 Culturing and
Differentiation of Cells
μ
g/mL poly-
L
-ornithine
and 3 h with 4
g/mL human fi bronectin. hmNPCs are pas-
saged when approaching a confl uence of 80-90 %. Cells are
detached with accutase, harvested with PBS, centrifuged at
600 ×
g
, at 20 °C, 10 min, and plated at the density of 30,000
cells/cm
2
.
μ
