This document discusses in vitro transcription and fluorescent in situ hybridization (FISH) techniques for visualizing gene expression in tissue samples. It describes the process of designing gene-specific primers, amplifying the gene of interest via PCR, synthesizing fluorescently-labeled RNA probes from the PCR product, hybridizing the probes to tissue samples, and using fluorescence microscopy to visualize where in the tissue the gene is expressed at a cellular level. The document provides technical details on each step of the process and discusses ways to optimize and troubleshoot the technique.

1. Tando MADUNA
INCI-CNRS UPR-3212 (Molecular Determinants of Pain)
Effect of neuropeptides on the development and plasticity of the nociceptive
system
(DoctoNeuro Neurotech Seminar Series 2014)

2. Background
 Gall and Pardue, 1969
 John et al, 1969
 Discovery of radioactive isotopes!
▪ Limited shelf-life
▪ Tedious procedure for autoradiography
▪ Limited spatial resolution
▪ Single-label visualization per specimen

3. in vitro transcription
+
hybridization
Tissue-specific expression
Post-transcriptional modifications
Post-translational modifications
Rat brain
(coronal)
Mouse
embryo

4. Digoxigenin-labeling Biotin-labeling Fluorescence
(fluoroscein)

5. AxioVision Rel4.7.1
(Carl Zeiss).
Stereomicroscopy
SteREO Discovery V12
AxioCam MRc
(Carl Zeiss)
Digoxigenin-labeling Visualization1 2 Qualitative Imaging
and Analysis
3

6. Probe synthesis
Sample preparation
ISH

7. PRIMER DESIGN
 Identify cds
(http://www.ncbi.nlm.nih.gov)
 Nucleotide BLAST cds
(http://blast.ncbi.nlm.nih.gov/Blast.cgi)
 Design specific primer sets
 (http://primer3.ut.ee/)
 Primer size, etc
 PCR conditions, i.e. Tm
 Primer-BLAST
 Verify the PCR product NM#
 Should also verify specificity of primer for your
specific gene (forward and reverse)
Forward and reverse primer
sequences for genes of interest
(GOIs), Vpac1 and Pax6

8. VERIFY PRIMERS AND SEQUENCE OF INTEREST (PCR PRODUCT)
 PCR to verify the size of the PCR product ≤800bp
Ladder Chk1 Vpac1 Pax6Denaturation
95°C
Annealing
55-65°C
Extension
72°C
RNA cDNA (sequence of interest = amplified PCR product)Reverse
Transcription
1 to 1,2% agarose gel in 1 x TAE Buffer
(Gel DOC™ EZ Imager, Bio-Rad)
Polymerase Chain Reaction (PCR) with previously designed specific primer sets
Verification of primers with PCR product

9.  Amplify the PCR product
 Plasmid vector
 and Competent Bacteria
VERIFY PRIMERS AND SEQUENCE OF INTEREST (PCR PRODUCT)
1
2

10. VERIFY PRIMERS AND SEQUENCE OF INTEREST (PCR PRODUCT)
Ligation of PCR product into multiple
cloning site of plasmid
2
1
DH5α
or
TOP10F
2 Transformation of plasmid
in competent bacteria
Resuspension
Lysis
Neutralization
Miniprep Kit
Plasmid extraction
3
multiple cloning site

11. Ladder Tbr1 Vpac1 Mcph1 Ladder Tbr1 Vpac1 Mcph1 Ladder Vpac1Chk1
Presence of PCR product by EcoRI digestion Orientation of synthesis Linearization of plasmid
1 to 1,2% agarose gel in 1 x TAE Buffer
(Gel DOC™ EZ Imager, Bio-Rad)
Restriction sites
Restriction map of the PCR product
(http://www.restrictionmapper.org/)

12. Post-transcriptional modifications
Post-translational modifications
Dig-UTP
Sp6 or T7 promotor + Buffer
RNAse Inhibitor
DTT (Cleland’s reagent)
Linearized plasmid (excised from
agarose gel shown previously)
DNAse treatment
Precipitate (NaAc + EtOH)
Day 1 of in vitro
transcription
Transcription of complementary
GOI sequence from linearized
plasmid with Dig-labelled UTP
and other unlabelled nucleotides

13. Resuspend pellet (plasmid) in
EtOH
Measure concentration of the
probe in DNA or RNA gel
(see next slide)
Day 2 of in vitro
transcription
Post-transcriptional modifications
Post-translational modifications

14. Resuspend pellet
(complementary RNA
probe) in EtOH
Measure concentration by
measuring relative band
intensity of smudge on
DNA gel
28S
18S
Quantitative?!
Spectrophotometry
Agarose (DNA) gel MOPS (RNA) gel
Measure single
stranded nucleic acid
concentration

15. Probe synthesis
Sample preparation
ISH

16. Pregnant mice
or rats
Cervical
dislocation
Whole-mount in
situ Hybridization
(WISH)
in situ Hybridization
(ISH)
Intracardiac perfusion of postnatal tissue to assay
Fixation in PFA +
Dehydration
Postnatal mice
or rats

17. 3’
3’5’
DIG-labelled
probe
5’
S
5’ 3’
5’3’
+ NBT/BCIP
Chromogenic Revelation
Non-specific hybridization Specific hybridization
AS
S- sense; AS- antisense; AP- alkaline phosphate
Image Analysis Discovery
V12
+
AxioCam MRc AxioVision
Rel.4.7.1 (Carl Zeiss)

18. Rehydration series
Tissue permeabilization
(Proteinase K)
Postfixation
Prehybridization
Formamide
SSC
Triton X100
Chaps
EDTA
Blocking powder
Heparin
Yeast RNA
Hybridization
Formamide
SSC
Triton X100
Chaps
EDTA
Blocking powder
Heparin
Yeast RNA
Dig-Probe
Wash series
(Form., SSC, detergents…)
Block for non-specific Ab
binding
Primary antibody
incubation
(anti-DIG-AP)
Wash series with
levamisol
BCIP/NBT
revelation
NBT - nitro-blue tetrazolium; BCIP - 5-bromo-4-chloro-3'-indolyphosphate
BCIP
NBT

19. Rehydration series
Tissue permeabilization
(Proteinase K)
Postfixation
Prehybridization
Hybridization
Wash series
(Form., SSC, detergents…)
Block for non-specific Ab
binding
Primary antibody
incubation
(anti-DIG-AP)
Wash series with
levamisol
NBT - nitro-blue tetrazolium; BCIP - 5-bromo-4-chloro-3'-indolyphosphate
BCIP
NBT
Dehydration series
Mount in xylene
AS S
BCIP/NBT
revelation
AS S

20.  Tissue permeabilization
 Temperature; Concentration of PK
 Pre- and Hybridization
 Temperature; Duration
 SSC concentration for stringency
 Antibody concentration
 Revelation duration
 Probe concentration
 Optimize primers

21. Image Analysis
AxioVision Rel4.7.1 (Carl
Zeiss).
Stereomicroscopy
SteREO Discovery V12
AxioCam MRc
(Carl Zeiss)
Optical microscope
Mainly reflected light
Left & Right viewing for 3D
visualization
Highly qualitative
WT KO
Cryosection
Image postWISH slices
(Post)Counterlabelling

22. Rehydration series
Tissue permeabilization
(Proteinase K)
Postfixation
Prehybridization
Hybridization
Wash series
(Form., SSC, detergents…)
Block for non-specific Ab
binding
Primary antibody incubation
(anti-DIG-AP)
NBT - nitro-blue tetrazolium; BCIP - 5-bromo-4-chloro-3'-indolyphosphate
Postfix in 4% PFA
Wash in PBS
Blocking solution
1° antibody solution
with Triton X100
AS
S
Wash in PBS
Blocking solution
2° antibody solution
with Triton X100
DAPI wash
Aqeous mounting
ImmunohistochemistryISH

23. Brightfield for mRNA Alexa488 for GFAP Merge
ISH Post-ISH IHC

24.  Multiple colour ISH
 Multiple colour FISH
 Multiple colour ISH-FISH
 Revelation with NBT-BCIP + FITC-conjugated
probe

25.  The Atlas of Mouse Development, Kaufman 2010
 Nonradioactive In Situ Hybridization, Second Edition, 1996,Boehringer Manheim
 PCR Protocols:A guide to Methods and Applications, Innis et al, 1990
 Correira and Conlon, 2001
 Molecular Cloning: A Laboratory Manual, Sambrook et al, First toThird Edition…
Thank you for your attention!!!