We present a FISH-based method for detecting single- nucleotide variants (SNVs) in exons and introns on individual RNA transcripts with high efficiency. We used this method
to quantify allelic expression in cell populations and in single cells, and also to distinguish maternal from paternal chromosomes in single cells.
3. Human cells produce RNA
using two copies of a gene
Mutant Wild-type
Wild-type RNA
Mutant RNA
Transcription
Sites
Nucleus
Cytoplasm
Heterozygotic cell
4. RNA FISH probes directly
detect RNA in single cells
RNA
FISH: Fluorescence in situ Hybridization
5. How do we distinguish
chromosomes and their RNA?
Chr. 19EEF2 mRNAEEF2 intron
Levesque and Raj, Nature Methods doi:10.1038/nmeth.2372
6. Our probes are not sensitive to
single nucleotide differences
RNA
7. Our probes are not sensitive to
single nucleotide differences
RNA
8. Our probes are not sensitive to
single nucleotide differences
RNA
9. A G U A G U C C G G G A A A U C G A U C C A G A C A A C U G U A G G U A A C U C
Masked probes provide specificity
thru a short “toehold” sequence
C T T T A G C T A G G T C T G T T G
G A T C C A G A C A A C
probe
mask
toehold dye
RNA target
10. A G U A G U C C G G G A A A U C G A U C C A G A C A A C U G U A G G U A A C U C
C
T
A
G
G
T
C
T
G
T
T
G
G
A
T
C
C
A
G
A
C
A
A
C
C T T T A G
Masked probes bind through
DNA strand displacement
probe
m
ask
dye RNA target
toehold
11. C T T T A G C T A G G T C T G T T G
G A T C C A G A C A A C
A G U A G U C C G G G A A A U C G A U C C A G A C A A C U G U A G G U A A C U C
Masked probes bind through
DNA strand displacement
probe
mask
dye
RNA target
toehold
12. A G U A G U C C G G G A A A C C G A U C C A G A C A A C U G U A G G U A A C U C
C
T
A
G
G
T
C
T
G
T
T
G
G
A
T
C
C
A
G
A
C
A
A
C
C T T T A G
Competing masked probes
distinguish single nucleotide variants
probe
m
ask
toehold
RNA target
dye
13. A G U A G U C C G G G A A A C C G A U C C A G A C A A C U G U A G G U A A C U C
C
T
A
G
G
T
C
T
G
T
T
G
G
A
T
C
C
A
G
A
C
A
A
C
C T T T G G
RNA target
probe
m
ask
toehold
C T T T A G C T A G G T C T G T T G
G A T C C A G A C A A C
dye
Competing masked probes
distinguish single nucleotide variants
14. C T T T G G C T A G G T C T G T T G
A G U A G U C C G G G A A A C C G A U C C A G A C A A C U G U A G G U A A C U C
toehold
RNA target
G A T C C A G A C A A C
mask
probe dye
C T T T A G C T A G G T C T G T T G
G A T C C A G A C A A C
Competing masked probes
distinguish single nucleotide variants
18. Single oligo detection assay takes
advantage of co-localization
Levesque et al, Nature Methods, doi:10.1038/nmeth.2589
19. Single oligo detection assay takes
advantage of co-localization
Levesque et al, Nature Methods, doi:10.1038/nmeth.2589
20. SNP FISH clearly shows
genotype of melanoma cell lines
Levesque et al, Nature Methods, doi:10.1038/nmeth.2589
21. SNP FISH shows mRNA allelic
imbalance in the cell population
Levesque et al, Nature Methods, doi:10.1038/nmeth.2589
22. SNP FISH shows mRNA allelic
imbalance in single-cells
Levesque et al, Nature Methods, doi:10.1038/nmeth.2589
23. SNP FISH shows mRNA allelic
imbalance in single-cells
p = 0.00017
Levesque et al, Nature Methods, doi:10.1038/nmeth.2589
24. SNP FISH shows mRNA allelic
imbalance in single-cells
p = 0.083
Levesque et al, Nature Methods, doi:10.1038/nmeth.2589
25. SNP FISH shows mRNA allelic
imbalance in single-cells
p = 0.83
Levesque et al, Nature Methods, doi:10.1038/nmeth.2589
26. SNP FISH + iceFISH distinguishes
maternal from paternal chromosome
Levesque et al, Nature Methods, doi:10.1038/nmeth.2589
Levesque and Raj, Nature Methods doi:10.1038/nmeth.2372
27. SNP FISH + iceFISH distinguishes
maternal from paternal chromosome
Levesque et al, Nature Methods, doi:10.1038/nmeth.2589