Article thumbnail

Minimizing off-target signals in RNA fluorescent in situ hybridization

By Aaron Arvey, Anita Hermann, Cheryl C. Hsia, Eugene Ie, Yoav Freund and William McGinnis


Fluorescent in situ hybridization (FISH) techniques are becoming extremely sensitive, to the point where individual RNA or DNA molecules can be detected with small probes. At this level of sensitivity, the elimination of ‘off-target’ hybridization is of crucial importance, but typical probes used for RNA and DNA FISH contain sequences repeated elsewhere in the genome. We find that very short (e.g. 20 nt) perfect repeated sequences within much longer probes (e.g. 350–1500 nt) can produce significant off-target signals. The extent of noise is surprising given the long length of the probes and the short length of non-specific regions. When we removed the small regions of repeated sequence from either short or long probes, we find that the signal-to-noise ratio is increased by orders of magnitude, putting us in a regime where fluorescent signals can be considered to be a quantitative measure of target transcript numbers. As the majority of genes in complex organisms contain repeated k-mers, we provide genome-wide annotations of k-mer-uniqueness at∼aarvey/repeatmap

Topics: Methods Online
Publisher: Oxford University Press
OAI identifier:
Provided by: PubMed Central

To submit an update or takedown request for this paper, please submit an Update/Correction/Removal Request.

Suggested articles


  1. (2003). Annotating large genomes with exact word matches.
  2. (2000). Assessment of the sensitivity and specificity of oligonucleotide (50mer) microarrays.
  3. (1990). Basic local alignment search tool.
  4. (1991). Control of the expression of the bithorax complex genes abdominal-A and abdominal-B by cis-regulatory regions in Drosophila embryos.
  5. (2009). Counting Hox transcripts within single cells in fixed Drosophila embryos: evidence for transcriptional bursting.
  6. (1984). Detection of mRNAs in sea urchin embryos by in situ hybridization using asymmetric RNA probes.
  7. (1981). Detection of poly A+ RNA in sea urchin eggs and embryos by quantitative in situ hybridization.
  8. (1978). Detection of viral sequences of low reiteration frequency by in situ hybridization.
  9. (1987). Distribution of the Sex combs reduced gene products in Drosophila melanogaster.
  10. (2005). Empirical establishment of oligonucleotide probe design criteria.
  11. (2005). Expression pattern of Wnt signaling components in the adult intestine.
  12. (2003). Fluorescence in situ hybridization: past, present and future.
  13. (2008). Fluorescent in situ hybridization protocols in Drosophila embryos and tissues.
  14. (2009). FlyBase: enhancing Drosophila Gene Ontology annotations.
  15. (2008). Imaging individual mRNA molecules using multiple singly labeled probes.
  16. (2004). Multiplex detection of RNA expression in Drosophila embryos.
  17. (2006). PROBER: oligonucleotide FISH probe design software.
  18. (2006). Short blocks from the noncoding parts of the human genome have instances within nearly all known genes and relate to biological processes.
  19. (2008). Single-cell transcription site activation predicts chemotherapy response in human colorectal tumors.
  20. (1993). Suffix arrays: A new method for on-line string searches.
  21. (2003). Visualization of single molecules of mRNA in situ.
  22. (1998). Visualization of single RNA transcripts in situ.