Ra SH Rapid Subtraction Hybridization Identifies differentially expressed

Ra. SH: Rapid Subtraction Hybridization • Identifies differentially expressed genes without prior knowledge of their functional or biochemical characteristics • Simplifies the process of c. DNA subtraction and requires small amounts of RNA • Selects genes YES or NOT expressed in specific conditions • Low cost and short time procedure

OBTENTION OF FRAGMENTS OF c. DNA(ds) DIGESTED WITH MBO I Extration RNA infected with NLS and NLS-cd st ge Cd Cd di di S NL Φ (ds)c. DNA syntesis NL S ge ste ed d Reaction of restriction 600 bp Actin Φ NLS CD Fragments of 500 to 200 bp

ADAPTERS LIGATION AND OBTENTION OF DRIVER 5’GATC Φ CTAG 5’ XDPN-14 5’CTGATCACTCGAGA 5’GATC 3’ TGAGCTCTCTAG XDPN-12 XDPN-18 NLS Cd XDPN-12 GATCTCTCGAGT 3’ ’ CTAG 5’ AGACTCACTAGTC 5 XDPN-14 XDPN-18 Precipitation of PCR large scale 20 reactions DRIVER

DIGESTION OF TESTER DRIVER Cd NLS Digestion (Xho. I) TESTER- cohesive ends DRIVER- no cohesive ends

SUBTRACTION TESTER DRIVER Do NOT enter plasmid Enter plasmid Do NOT enter plasmid Restrition site Xho. I allows binding of selected fragments to p. Ze. Ro

TWO CONDITIONS OF SUBTRACTION NLS TESTER Genes expressed when nuclear calcium is buffered ↓ [Ca 2+]nuc → ↓ proliferation Cd TESTER Genes expressed when nuclear calcium is present ↑ [Ca 2+]nuc → ↑ proliferation

RESULTS

GENES EXPRESSED IN THE ABSENCE Ca 2+ • Tubulin, gamma 1 (TUBG 1) • Mob 4 B protein • Splicing factor 3 b • Ras homolog gene family, member A (RHOA) • ARID 1 A • Alanyl-t. RNA synthetase (AARS) • RTN 4 isoform G

RTN 4 ISOFORM G • Reticulons (RTN) are a group of integral membrane proteins that are involved in various neurodegenerative disease (Zhou X. et. al. , Cell. Mol. Life Sci. , 2006) • RTN 4 (Nogo) has been intesively investigated as a critical inhibitory controlling axonal growth and regeneration (Reichardt P. et al. , Trans. R. Soc. , 2006) • RTN 4 interacts with neurotrophin receptor p 75 NTR forming a complex that can inhibite neurite outgrowth (He Z. et al. , Nature, 2002) • p 75 NTR expression by hepatic stellate cells is necessary for their differentiation to repair-supporting, HGF-secreting cells, which in turn can promote hepatocyte proliferation (Akassoglou K. et al. , Science, 2007)

RTN 4 GENE EXPRESSION – SAGE Normal Brain Lung Pancreas Cancer brain Lung Pancreas Peritonium Muscle Breast cell subtupes ES cells Peritonium Breast cell subtupes

RTN 4 ISOFORMS AND RT-PCR TARGET 397 bp