RaSH: Rapid Subtraction Hybridization
• Identifies differentially expressed genes without prior
knowledge of their functional or biochemical characteristics
• 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
OBTENTION OF FRAGMENTS OF cDNA(ds)
DIGESTED WITH MBO I
Extration RNA infected with
NLS and NLS-cd
Reaction of restriction
Φ
(ds)cDNA syntesis
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
PCR large scale
20 reactions
DRIVER
DIGESTION OF TESTER
DRIVER
Cd
NLS
Digestion
(XhoI)
TESTER
TESTER- cohesive ends
DRIVER- no cohesive ends
SUBTRACTION
TESTER
DRIVER
Do NOT
enter
plasmid
Enter plasmid
Do NOT
enter
plasmid
Restrition site XhoI allows
binding of selected fragments
to pZeRo
TWO CONDITIONS OF SUBTRACTION
NLS TESTER
Cd TESTER
Genes expressed when
nuclear calcium is buffered
↓ [Ca2+]nuc → ↓ proliferation
Genes expressed when
nuclear calcium is present
↑ [Ca2+]nuc → ↑ proliferation
RESULTS
GENES EXPRESSED IN THE ABSENCE Ca2+
• 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
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)
RTN4 GENE EXPRESSION – SAGE
Normal
Brain
Lung
Pancreas
Cancer
brain
Lung
Pancreas
Peritonium
Muscle
Muscle
Breast cell subtupes
ES cells
Peritonium
Breast cell subtupes
RTN4 ISOFORMS AND RT-PCR TARGET
397 bp