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RaSH: R a pid S ubtraction H ybridization

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. RaSH: R a pid S ubtraction H ybridization.

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RaSH: R a pid S ubtraction H ybridization

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  1. 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 RaSH:Rapid Subtraction Hybridization • Identifies differentially expressed genes without prior knowledge of their functional or biochemical characteristics

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

  3. Φ NLS Cd 5’GATC CTAG 5’ XDPN-14 XDPN-12 5’CTGATCACTCGAGA GATCTCTCGAGT3’ AGACTCACTAGTC 5’ 5’GATC 3’TGAGCTCTCTAG XDPN-14 XDPN-12 CTAG 5’ Precipitation of PCR XDPN-18 XDPN-18 PCR large scale DRIVER 20 reactions ADAPTERS LIGATION AND OBTENTION OF DRIVER

  4. Digestion (XhoI) TESTER TESTER- cohesive ends DRIVER- no cohesive ends DIGESTION OF TESTER DRIVER Cd NLS

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

  6. Genes expressed when nuclear calcium is buffered NLSTESTER ↓ [Ca2+]nuc→ ↓ proliferation Genes expressed when nuclear calcium is present Cd TESTER ↑ [Ca2+]nuc→ ↑ proliferation TWO CONDITIONS OF SUBTRACTION

  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 Ca2+

  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 p75NTR forming a complex that can inhibite neurite outgrowth (He Z. et al., Nature, 2002) • p75NTR 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 Normal Cancer brain Brain Lung Lung Pancreas Pancreas Peritonium Peritonium Muscle Muscle Breast cell subtupes Breast cell subtupes ES cells

  11. RTN4 ISOFORMS AND RT-PCR TARGET 397 bp

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