Transcriptional Characterization of Glioma Neural Stem Cells Diva ...

6.5 Large-scale qRT-PCR Validation Results
for expression profiles of individual genes was 0.91 and the differential expres-
sion calls were corroborated for all 82 genes. Figure 6.12 shows the relationship
between the qRT-PCR and the Tag-seq measurements in three different panels
for four different genes that were picked to highlight the dynamic range of the
qRT-PCR and the Tag-seq platforms: the matrix metallopeptidase is a mem-
brane protein involved in the breakdown of the ECM and a known oncogene in
glioblastoma primary tumours [183]; the damage-inducible transcript DDIT3
is induced in response to a range of compounds that sensitises glioma cells
to apoptosis [216]; MYL9 encodes for one of the myosin light chains and its
link with glioblastoma has yet to be established (possibly acting via regulation
of the cell migration process); the mannosidase gene MAN1C1 is one of the
enzymes that hydrolyse the mannose sugar but has to yet be implicated in
glioblastoma - although its sister gene MAN2C1 has been associated with a
reduction of PTEN functionality in prostate cancer [190]. In panel a of figure
6.12 the high overall correlation between the two platforms is highlighted by
the four data points that lie nearly on a straight line (R = 0.91); panel b shows
the correlation values computed as the correlation between normalised Ct val-
ues and tag counts across the five GNS cell lines (table of correlation values
available in Appendix A.4); panel c shows the mean fold-change between the
genes expressed in the GNS and NS cell lines as measured in each of the two
platforms plotted so that the influence of outliers can be reduced thanks to
the averaging.
Across the entire panel of cell lines, 29 of the 82 genes showed statistically
significant differences between GNS and NS lines at an FDR of 5% (Fig 6.13).
This set of 29 genes distinguishes GNS from NS lines and may, therefore, have
broad relevance for elucidating tumourigenic properties of GNS cells. Figure
6.14 shows a histogram for each of the 29 genes found to be relevant in distin-
guishing GNS from NS cell lines. The height of the histogram bar represents
the expression level measured via qRT-PCR for that gene and its designated
probe (see title of each histogram for probe’s name). Each expression level is
calculated as the arithmetic mean across all biological replicates for each cell
line. The histogram bar colours comply to the ones selected for GNS and NS
cell lines in the bar displayed at the top of fig 6.14, distinguishing the assayed
GNS and NS cell lines in two separate groups along the x-axis.
137