Transcriptional Characterization of Glioma Neural Stem Cells Diva ...

5.4 Quantitative Real Time-PCR Validation Methods
PCR. This gene set comprises 82 validation targets from Tag-seq analysis, eight
glioma and developmental markers, and three endogenous control genes - 18S
ribosomal RNA, TUBB and NDUFB10. The 18S gene was chosen because
used by ABI as a manufacturing control, while TUBB and NDUFB10 were
selected because they are present in our Tag-seq dataset and show low varia-
tion of expression across GNS and NS cell lines in an independent microarray
expression dataset [404]. The 93 genes were interrogated using 96 different
TaqMan assays (three of the validation targets required two different primer
and probe sets to cover all known transcript isoforms matching differentially
expressed tags). cDNA was generated using SuperScript III (Invitrogen) and
real-time PCR carried out using TaqMan fast universal PCR master mix. The
absence of a no-template control (NTC) amplification (horizontal slope) en-
sured that random contamination and reagent contamination were not affect-
ing our samples. In complying with the minimum information for publication
of quantitative real-time PCR experiments (MIQE) [78], a full assay list with
raw and normalised threshold cycle (Ct) values is provided in Appendix A.3.
The data analysis was performed using the R package HTqPCR [131], which
handles high-throughput qPCR data with a focus on data from Taqman low
density arrays. Ct values were normalised to the average of the three control
genes and potential outliers identified and filtered out using the plotCtBoxes
function (Fig 5.2). Figure 5.3 shows the effect of the normalisation method
Figure 5.2: Boxplot of normalised Ct values identifies outliers (empty circles).
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