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