visualizing snvs to quantify allele-specific expression in single cells
TRANSCRIPT
Visualizing SNVs to quantify allele-specific
expression in single cells
Marshall J Levesque, Paul Ginart, Yichen Wei, Arjun RajSystems Biology Lab, UPenn Bioengineering
Nature Methods (2013) doi:10.1038/nmeth.2589
Paternal RNA
Maternal RNA
Transcription Sites
Nucleus
Cytoplasm
Heterozygotic cellMaternal Paternal
Human cells produce RNA using two copies of a gene
Human cells produce RNA using two copies of a gene
Mutant Wild-type
Wild-type RNA
Mutant RNA
Transcription Sites
Nucleus
Cytoplasm
Heterozygotic cell
How do we distinguish chromosomes and their RNA?
Chr. 19EEF2 mRNAEEF2 intronLevesque and Raj, Nature Methods doi:10.1038/nmeth.2372
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 GG A T C C A G A C A A C
probe
mask
toehold dye
RNA target
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
CTAGGTCTGTTG
GATCCAGACAAC
C T T T A G
Masked probes bind through DNA strand displacement
probe
mask
dye
RNA target
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
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
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
CTAGGTCTGTTG
GATCCAGACAAC
C T T T A G
Competing masked probes distinguish single nucleotide variants
probe
mask
toehold
RNA target
dye
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
CTAGGTCTGTTG
GATCCAGACAAC
C T T T G G
RNA target
probe
mask
toehold
C T T T A G C T A G G T C T G T T GG A T C C A G A C A A C
dye
Competing masked probes distinguish single nucleotide variants
C T T T G G C T A G G T C T G T T GA 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 GG A T C C A G A C A A C
Competing masked probes distinguish single nucleotide variants
Single oligo detection assay takes advantage of co-localization
Levesque et al, Nature Methods, doi:10.1038/nmeth.2589
Single oligo detection assay takes advantage of co-localization
Levesque et al, Nature Methods, doi:10.1038/nmeth.2589
SNP FISH clearly shows genotype of melanoma cell lines
Levesque et al, Nature Methods, doi:10.1038/nmeth.2589
SNP FISH shows mRNA allelic imbalance in the cell population
Levesque et al, Nature Methods, doi:10.1038/nmeth.2589
SNP FISH shows mRNA allelic imbalance in single-cells
Levesque et al, Nature Methods, doi:10.1038/nmeth.2589
SNP FISH shows mRNA allelic imbalance in single-cells
p = 0.00017
Levesque et al, Nature Methods, doi:10.1038/nmeth.2589
SNP FISH shows mRNA allelic imbalance in single-cells
p = 0.083
Levesque et al, Nature Methods, doi:10.1038/nmeth.2589
SNP FISH shows mRNA allelic imbalance in single-cells
p = 0.83
Levesque et al, Nature Methods, doi:10.1038/nmeth.2589
SNP FISH + iceFISH distinguishes maternal from paternal chromosome
Levesque et al, Nature Methods, doi:10.1038/nmeth.2589Levesque and Raj, Nature Methods doi:10.1038/nmeth.2372
SNP FISH + iceFISH distinguishes maternal from paternal chromosome
Levesque et al, Nature Methods, doi:10.1038/nmeth.2589