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

2. Extration RNA infected with NLS and NLS-cd (ds)cDNA syntesis Actin Φ NLSCD OBTENTION OF FRAGMENTS OF cDNA(ds) DIGESTED WITH MBO I Fragments of 500 to 200 bp 600 bp Φ Cd Cd digested NLS NLS digested Reaction of restriction

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

4. DIGESTION OF TESTER NLSCd DRIVERTESTER Digestion (XhoI) TESTER- cohesive ends DRIVER- no cohesive ends

5. SUBTRACTION Do NOT enter plasmid TESTERDRIVER Do NOT enter plasmid Enter plasmid Restrition site XhoI allows binding of selected fragments to pZeRo

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

7. RESULTS

8. Tubulin, gamma 1 (TUBG1) Mob4B protein Splicing factor 3b Ras homolog gene family, member A (RHOA) ARID 1A Alanyl-tRNA synthetase (AARS) RTN4 isoform G GENES EXPRESSED IN THE ABSENCE Ca 2+

9. RTN4 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) RTN4 (Nogo) has been intesively investigated as a critical inhibitory controlling axonal growth and regeneration (Reichardt P. et al., Trans. R. Soc., 2006) RTN4 interacts with neurotrophin receptor p75 NTR forming a complex that can inhibite neurite outgrowth (He Z. et al., Nature, 2002) p75 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)

10. RTN4 GENE EXPRESSION – SAGE NormalCancer Muscle ES cells Breast cell subtupes Peritonium Pancreas Brain brain Lung Breast cell subtupes

11. RTN4 ISOFORMS AND RT-PCR TARGET 397 bp