Preparation of samples for expression array

In order to understand the transcriptional repression role of Sox2 in hNSCs, Sox2 and Sox2 LQY/AAA expression plasmid were introduced into hNSCs. In theory, the faster a gene’s response to a transcription factor, the more likely the effect of the transcription factor function is direct. Hence, in order to obtain data that reflect possible Sox2 directly regulated genes, it was considered better to collect cells at the earliest time point of Sox2 expression.

To monitor the expression level of an exogenous gene in hNSCs, a GFP expression vector was co-transfected into hNSC with Sox2 and the cells expressing GFP were observed (Figure 5.2.1). The results show that exogenous gene expression increased dramatically 14 hours after transfection (Figure 5.2.2). Therefore, this time point was used to collect hNSC for the expression array analysis. Since the transfection efficiency of hNSCs was always below 100%, non-transfected cells still exist within the transfected cell population. The observation under the fluorescent microscope showed approximately 40% of the cell population were non-transfected, therefore the noise from the non- transfected cells would have a significant potential affect on the expression array data. In order to avoid the noise from these non-transfected cells, Fluorescence Activated Cell Sorting (FACS) was carried out on the cells 14 hours after transfection.

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Figure 5.2.1 Timecourse of GFP expression in transfected hNSCs

hNSCs were transfected with GFP/Sox2 and the green and non-green fluorescent cells were observed at different time points (8-24 hours) and the cells counted.

Figure 5.2.2 Flow chart of Affymetrix experiment

hNSCs were collected 14 hours after transfection with either GFP alone, GFP with wild type Sox2 or GFP with Sox2 LQY/AAA. Fluoresence Activated Cell Sorting (FACS) was carried out to sort out the untransfected (GFP negative) cells. The GFP positive cells were collected and the total RNA extracted immediately. Total RNA was purified and applied to the Affymetrix expression array system.

During the electroporation procedure, it is very likely that cells take up GFP and Sox2 plasmid DNA simultaneously. Previous data in our lab suggested more than 90% co-transfection. Therefore, the cells expressing GFP are very likely to also express exogenous Sox2. According to this hypothesis, the Sox2

126 expressing cells could be sorted through the expression of GFP. FACS allowed the separation of the healthy and GFP positive cells from a group of cells (Figure 5.2.3).

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Figure 5.2.3 FASC sorting charts

FACS detected the volume of cells by light scatter intensity (Figure 5.2.4 A). The healthy cells in region A were selected and the green strength of cells was detected (B). The fluorescence intensity of GFP expressing cells was measured and the cells which had higher fluorescence intensity (higher than 10 units) was shown in panel C. Cells which expressed fluorescence higher than 10 units were selected (region C in panel C) as the green positive cells whereas the low green level cells were selected as green negative (region F in panel C). Panel E-G show the distribution of fluorescence in GFP, GFP/Sox2 or GFP/Sox2LQY/AAA transfected cells. The cells in R1 region were collected. Panel H shows the relative graph of fluorescence intensity with cell numbers. The region R1 in panel E-G is marked as M1 in panel H.

A. B.

C. D.

E. F.

128 The FACS technique detected the volume of cells by light scatter intensity (Figure 5.2.3 A). The parameters of every cell were shown as a dot on the SS/FS cytogram. More than 90% of cells were healthy in the transfected hNSCs population (Figure 5.2.3 D). The healthy cells in region A were selected and the green fluorescence level of cells were detected (Figure 5.2.3 B, C). Cells expressing more than 10 units of fluorescence were selected (region C in Figure 5.2.3 C, approximate 27% of region A selected cells) Figure 5.2.3 E-G shows the distribution of fluorescence in GFP, GFP/Sox2 or GFP/Sox2LQY/AAA transfected cells. The cells in R1 region were collected and the relative graph of fluorescence intensity with cell numbers is showed in Figure 5.2.3 H.

In order to avoid the degradation of RNA, cells were lysed immediately in TRI reagent. The total RNA of GFP positive cells was purified using RNeasy® Mini column (Qiagen). The quality of the total RNA was checked using a 2100 BioAnalizer (Agilent) (Figure 5.2.4). The RNA integrity number of all three sets of total RNA (GFP, GFP/Sox2 and GFP/ Sox2 LQY/AAA) was 10 which indicated the quality of the total RNA was good enough.

The Affymetrix GeneChip® Human Genome U133 plus2 array was chosen to detect the expression profile in this chapter. There are up to 1.3 million different oligonucleotide probes attached on the chip which offers a rapid and thorough assay of human gene expression. Figure 5.2.2 illustrates the experimental strategy to determine the effect of Sox2 overexpression in hNSC using Affymetrix expression arrays. Further analysis of the expression array data will be discussed in the next section.

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Figure 5.2.4 RNA quality checking

The total RNA of GFP, GFP/Sox2 and GFP/ Sox2 LQY/AAA transfeced hNSCs were analysed by 2100 Bio Analizer (Aglient). Simulated electrophoresis images are presented on the right.The ladder graph shows the marker peaks as control. The 18s and 28s peaks are shown in graphs from GFP, Sox2 and Sox2 LQY/AAA transfected cells.

Figure 5.2.5 Diagr The total RNA f Affymetrix GeneC dT primers was ca was prepared from fragmented before automated washing staining, the chip w probe cells and com

gram of Affymetrix GeneChip® Human Genome U1

from different experiments of hNSCs was subjecte eChip® Human Genome U133 plus2 arrays. Reverse tr carried out on the purified mRNA to generate cDNA rom the cDNA using an in vitro transcription met re hybridization. After 16 hours of hybridization, th ing and staining in a fluidics station. Immediately follo was scanned using the Affymetrix Microarray Suite. T omputes an intensity for each cell. (http://www.dkfz.de/

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133 plus2 array. cted to analysis by the transcription using poly A. Biotin-labeled cRNA ethod. The cRNA was the chip underwent an llowing the washing and The software defines the

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