Whole-mount in situ Hybridization
Dec 19, 2015
Whole-mount in situ Hybridization
Zebrafish Development
Function?
Over-expression knockdown
DNA: using tissue specific promoter
RNA: in vitro transcription
Morpholino
Dominant negative form
Antagonist
Whole mount in situ hybridization
Whole mount in situ hybridization
Digoxigenin (DIG)
• DIG is a steroid hapten from Digitalis lanata (毛地黃 )
Structure of DIG-Modified Nucleotides Coupled via Alkali-Stable Ether Bond
Formula: C43H61N4O21P3Li4 Molecular Weight: 1090.7DIG-UTP (R1=OH, R2=OH) DIG-dUTP (R1=OH, R2=H) DIG-ddUTP (R1= H, R2=H)
Non-Radioactive RNA-Labeling
TA vector
SP6 T7Probe
HindIII EcoRIHindIII
Detection methods
• Chemiluminescent detection
CDP-star or CSPD
• Colorimetric detection
NBT/BCIP
• Fluorescent detection
• http://www.roche-applied-science.com/prodinfo_fst.htm?/DIG/dig_labeling03.htm
• http://zfin.org
• mRNA: FGF8 (Fibroblast Growth Factor 8)
• Probe: Sef (Similar Expression of Fgf gene)
(Endocrine Reviews 2005, 26: 63-77)
(Cytokine & Growth Factor Reviews 2005, 16: 139-149)
(Nature Cell Biology 2002, 4: 170-174)
(Mechanisms of Development 2002, 119: 91-108)
(Development 2002, 129: 917-933)