in situ hybridization techniques fribourg, march 06 2012 ... 2012.pdfapplications: few examples:...
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In situ hybridization techniques
Fribourg, March 06 2012
Franck Girard
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General comments - Applications
- Goal: Provides topological information on gene activity at the DNA/RNA level, through the specific detection of nucleic acids (RNA, DNA) in a morphologically preserved tissue
- Method: Based on the principle of pairing of complementary bases (A/T, G/C) Use of specific nucleotide probes (RNA, DNA, oligonucléotides), labeled (radioactivity, digoxygénine, biotine, fluorochromes)
- Applications: - cells in culture - tissue sections - chromosomes - whole organims (embryos)
- DNA microarray - Northern/Southern blot
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In situ hybridization to RNA – General comments
- Goal: determine which cells expressed a mRNA of interest
-Procedure: -probe preparation (Digoxygenine labeled antisense RNA probes) (radioactivity, fluorescent dye)
-tissue preparation (12µm brain cryosections) (paraffin sections, whole mount)
-in situ hybridization
-revelation/observation (anti-digoxygenine coupled to alkaline phosphatase) (microscope/fluorescence, autoradiography)
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Préparation of digoxygenine labeled RNA probes
Primer design Size probe: 300bp up to 1-2 Kbp (non homologous sequences)
cDNA clone Genomic DNA cDNA
PCR
RNA
Reverse transcription
In vitro transcription (digoxygenine)
DIG labeled riboprobes
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RNA mix (from tissue)
Reverse transcription
PCR (RT, nucleotides, Taqpol) SP6
T3
in vitro transcription (PCR, nucleotides, UTP-DIG, RNApol)
sense
antisense
Préparation of digoxygenine labeled RNA probes
Specific probe
Control (no hybridization)
Or cDNA
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In situ hybridization on brain sections - Protocole / RNA-DIG
- Brain dissection and freezing
- Cryosection (10 - 15 µm), sections stored at –80°C
- Fixation (4%paraformaldéhyde)
- Acétylation (triethanolamine / acetic anhydride) (reduces non spécific background)
- Perméabilisation (détergents, Protéinase K) (accessibilty to the target RNA)
- Préhybridation (55°-65°C) (formamide / SSC)
- Hybridization (55°-65°C, 16h) (formamide / SSC / denhardt / RNA yeast)
- Washes (55°-65°C, stringency %SSC from 2X to 0.1X)
- Anti-DIG antibody (coupled to alkaline phosphatase)
- Détection NBT/BCIP
- Mounting / Observation
(http://www.roche-applied-science.com/sis/lad/index.jsp)
Remarks: RNases gloves, autoclaved materials, DEPC
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In situ hybridization (RNA) - Principles. Détection
Target RNA Antisense-DIG
PA Substrate (NBT/BCIP)
Product (violet)
Anti-DIG
cytoplasm
nucleus
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Applications: few examples: tissue section (mouse)
Neurocalcin delta (calcium binding protein) Brain coronal section Probe RNA-DIG antisense
cortex
HPF
DG CA2
CA3
cortex HP
TH
HYP
Remark: Semi-quantitative method Distinguish no/low/medium/strong expression Quantitative levels: quantitative RT-PCR, DNA microarray
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Applications: few examples: tissue section (mouse)
Calbindin 1 (calcium binding protein) - Probe RNA-DIG antisense Coronal brain section / cerebellum kidney
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Applications: combining ISH and immunostaining (mouse brain)
Thrombospondin 4 (ISH) GFAP (immuno) merge
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Gene1 Fluo Gene2 Fluo
Applications: few examples: tissue section (mouse)
RNA1 Antisense-Fluo
RNA2 Antisense-Fluo
Fluorescent in situ hybridization on brain section
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Applications: few examples: embryos
Drosophila mouse (DIG)
Sea urchin (DIG) High throughput screen for developmentally regulated genes
(fluo X 7)
(DIG)
Whole mount in situ hybridization
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Applications: few examples: DNA microarrays, chromosomes, tissues
DNA microarray (fluo) Compare normal tissue/ Pathologic situation
chromosomes (fluo) Cytogenetics (Diagnosis): - mapping genes on chromosomes - Chromosomal abnormalities (duplication, translocation, deletion)
Pulmonary cells (DIG) Cytomégalovirus diagnosis
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Applications: Gene expression analysis in the mouse hypothalamus
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PV1 nucleus
Reticular nucleus of the thalamus
Dentate gyrus
CP
CTX
HY LHA
ZI
opt
VMH TU
STN
DMH
TH
V3
MEA BMA
BLA
int
opt
LHA
A B C
The Parvalbumin immunoreactive PV1 nucleus in the lateral hypothalamus
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Methods:
- Screen the Allen Atlas of mouse gene expression to find genes possibly co-expressed with Pvalb in the PV1 nucleus
- Analyse co-expression with Pvalb by in-situ hybridization on adjacent sections (mouse adult brain cryosections, DIG-labelled antisense RNA probes)
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Pvalb Vamp1 Vamp1 Pvalb
A’ A B B’
OT
LHA CTX
C’ C D D’
Adcyap1 Adcyap1 Pvalb Pvalb
Pvalb Slc17a6 Slc17a6 Pvalb
E’ E F F’
OT
Pvalb Slc17a6
PV1 3V
CTX MN
G H
Pvalb
G’
Slc17a6
H’
In situ hybridization on adjacent sections: co-expression with parvalbumin in the PV1 nucleus
76 candidate genes: 27 tested by ISH
19 co-expressed 8 not co-expressed
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Penk1 Pvalb
Tac1 Pvalb
Trh Pvalb
Cart Pvalb
Pdyn Pvalb
Tac2 Pvalb
A A’ B B’
C C’ D D’
E E’ F F’
In situ hybridization on adjacent sections: lack of major neuropeptides in the PV1 nucleus
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Conclusions:
- PV1 neurons are glutamatergic (express Slc17a6/Vglut2, lack GABA)
- Classical hypothalamic neuropeptides are not expressed in PV1 neurons, with the exception of Adcyap1/PACAP
- PV1 glutamatergic neurons (excitatory neurons) and GABAergic Pvalb neurons (inhibitory neurons in the cortex,hippocampus, thalamus) share a number of genes, including potassium and sodium channels