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

Reverse transcription with direct fluorescence labeling

A reverse transcription with direct labeling, meaning the incorporation of Cy3-dCTP or Cy5-dCTP during cDNA synthesis, was subsequently performed. 100 g total RNA, SuperScript II Reverse Transcriptase (Invitrogen) and gene-specific primer were used for this reaction. In order to remove the RNA at the end of the reaction, an alkaline RNA digest was performed at 65C.

Purification of the labeled cDNA

To avoid hybridization artifacts, the generated cDNA strands had to be separated from free fluorescence labeled nucleotides. By using YM30 columns (Millipore), it was possible to simultaneously remove the free nucleotides and attain a concentration of the cDNA.

Hybridization

For the hybridization reaction, the labeled cDNA was mixed with hybridization buffer (250 mM phosphate buffer / 1 x SSC / 1 mM EDTA / 4.8% SDS) and denatured for 3 min at 95C. The hybridization mix was then added to the hybridization area of the microarray and covered with a cover slip, avoiding air bubbles. The hybridization reaction was performed for 16 hours at 42C.

Detection of the hybridization signals

The detection and quantification of the hybridization events were done using a GMS 418 laser scanner (Affymetrix).

Fig. 1: Hybridization of a Rat Liver Chip printed on Eppendorf CreativeChip 3D Slide
Cy5 labeled cDNA: synthesized from 100 g total RNA from hepatocytes cultivated on collagen 1 w/o HGF (hepatocyte growth factor)
Cy3 labeled cDNA: synthesized from 100 g total RNA from hepatocytes
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