The experiment

We prepared cultures of NPCs from mice and differentiated these in vitro into more mature cells of the central nervous system: astrocytes, neurons, and oligodendrocytes (Figure 5.1) (1). Gene expression changes take place during the differentiation of NPCs and were analyzed with our cDNA microarray platform. We were mainly interested in early changes that take place during the transition from an undifferentiated cell type to a differentiated one. The hybridization scheme (Figure 5.1) depicts the cohybridization of cDNA derived from RNA of undifferentiated NPCs as a reference with targets from NPCs that were allowed to differentiate for 1, 2, or 4 days. Bioinformatics tools were used to normalize the obtained microarray data and to identify differentially expressed genes. This data was further subjected to a cluster analysis to deduce onset and dynamics of relevant gene expression changes (see also Chapters 19 and 20).

It is well known that cells change during cultivation. Therefore, we minimized the propagation time in vitro. However, this limits the amount of RNA available for analysis. To perform experiments with small amounts of

Figure 5.1.

Neural progenitor cell differentiation in vitro. The photographs depict morphological changes associated with adhesion, migration, and differentiation of neural progenitor cells within the first 4 days after induction of differentiation. Either neurotrophic growth factor BDNF or NT4 was added. Arrows indicate the microarray hybridization scheme, where RNA from differentiated cells was co-hybridized with RNA from undifferentiated cells as a reference. Scale bar equals 50 |m.

RNA, one can either amplify the starting material or the signal on the array. RNA can be amplified using a number of different protocols. They usually rely on the activity of reverse-transcriptase to generate cDNA, which serves as template for an RNA polymerase. The resulting RNA (aRNA) is then hybridized onto a microarray. A disadvantage is, however, that the amplification procedure potentially introduces a bias and therefore might change the original proportions of different transcripts that one actually wants to measure with a microarray. We decided to abstain from RNA amplification and instead applied a signal amplification method based on the 3DNA dendrimer technology (Genisphere, Hatfield, PA). This method relies on the specific hybridization of highly branched molecules called dendrimers to the array-bound samples. These structures carry multiple fluorescent labels like Cy3 or Cy5. Therefore, each probe on the array is visualized by many fluorescent molecules thereby increasing the signal intensity and facilitating the detection with the array scanner.

Our protocol describes the preparation of cDNA microarrays, the hybridization of cDNA samples derived from RNA of undifferentiated and differentiated NPCs to the arrays, signal amplification with 3DNA dendrimers, scanning of the arrays, processing of raw data, identification of differentially expressed genes, and a cluster analysis of relevant gene expression changes.

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