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An Optimal DNA Microarray Substrate for the Identification of Fetal ,,, and Somatic Liver Stem Cells of the Rat

erentiation possible. This will occur in connection with the optimization of culture conditions such as matrix, media and supplements. DNA microarrays specially developed for the identification of fetal hepatocytes will be used. The CreativeChip 3D Slides (Eppendorf) will be tested for its suitability as a base substrate for these DNA microarrays.

Material and methods

Cell culture

Fetal hepatocytes were cultivated under various conditions. The culture conditions result from the use of various growth factors (individually or combined) and an optimal matrix or "feeder" cells.

Microarray design and production

All genes relevant for the differentiation and growth of the liver have been identified from various databases. Oligonucleotides (50mers) were chosen from areas near the 3' ends of the mRNAs, which represent these genes sequence-specifically. The specificity of these oligonucleotides was tested with PCR*, Southern and Northern blots (not shown). The spotting of the oligonucleotides onto CreativeChip 3D Slides (Eppendorf) was performed using a GMS 417 array system (Affymetrix) according to manufacturer instructions.

Lyophilized oligonucleotides were dissolved in water and adjusted to a concentration of 100 M. Prior to printing, they were mixed 1:1 with spotting buffer (200 mM phosphate buffer (pH 9.0) / 10% Na2SO4 / 2 M betaine).

RNA isolation

Total RNA was isolated from the cells cultivated under various conditions with the RNeasy Kit (Qiagen). In order to obtain the cleanest and most DNA-free RNA possible, DNase digestion, phenol/chloroform/isoamyl alcohol purification and ethanol precipitation were performed.


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