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.

1. Visualizing SNVs to
quantify allele-specific
expression in single cells
Marshall J Levesque, Paul Ginart, Yichen Wei, Arjun Raj
Systems Biology Lab, UPenn Bioengineering
Nature Methods (2013) doi:10.1038/nmeth.2589

2. Paternal RNA
Maternal RNA
Transcription
Sites
Nucleus
Cytoplasm
Heterozygotic cell
Maternal Paternal
Human cells produce RNA
using two copies of a gene

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

15. RNA
Single oligos produce false positives
when bound to off-targets

16. RNA
Single oligos produce false positives
when bound to off-targets

17. Single oligos produce false positives
when bound to off-targets

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

28. Acknowledgements
Members of the Raj Lab
UPenn Bioengineering
Herlyn Lab
Wistar Institute
Biosearch Technologies