Visualizing SNVs to quantify allele-specific expression in single cells
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Visualizing SNVs to quantify allele-specific expression in single cells

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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 ...

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.

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Visualizing SNVs to quantify allele-specific expression in single cells Visualizing SNVs to quantify allele-specific expression in single cells Presentation Transcript

  • 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
  • Paternal RNA Maternal RNA Transcription Sites Nucleus Cytoplasm Heterozygotic cell Maternal 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
  • RNA FISH probes directly detect RNA in single cells RNA FISH: Fluorescence in situ Hybridization
  • How do we distinguish chromosomes and their RNA? Chr. 19EEF2 mRNAEEF2 intron Levesque and Raj, Nature Methods doi:10.1038/nmeth.2372
  • Our probes are not sensitive to single nucleotide differences RNA
  • Our probes are not sensitive to single nucleotide differences RNA
  • Our probes are not sensitive to single nucleotide differences RNA
  • 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
  • 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
  • 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 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
  • 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
  • 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
  • RNA Single oligos produce false positives when bound to off-targets
  • RNA Single oligos produce false positives when bound to off-targets
  • Single oligos produce false positives when bound to off-targets
  • 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.2589 Levesque 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
  • Acknowledgements Members of the Raj Lab UPenn Bioengineering Herlyn Lab Wistar Institute Biosearch Technologies