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In situ hybridization techniques
Fribourg, March 06 2012
Franck Girard
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
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)
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
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
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
In situ hybridization (RNA) - Principles. Détection
Target RNA Antisense-DIG
PA Substrate (NBT/BCIP)
Product (violet)
Anti-DIG
cytoplasm
nucleus
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
Applications: few examples: tissue section (mouse)
Calbindin 1 (calcium binding protein) - Probe RNA-DIG antisense Coronal brain section / cerebellum kidney
Applications: combining ISH and immunostaining (mouse brain)
Thrombospondin 4 (ISH) GFAP (immuno) merge
Gene1 Fluo Gene2 Fluo
Applications: few examples: tissue section (mouse)
RNA1 Antisense-Fluo
RNA2 Antisense-Fluo
Fluorescent in situ hybridization on brain section
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
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
Applications: Gene expression analysis in the mouse hypothalamus
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
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)
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
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
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