Minimizing off-target signals in RNA fluorescent in situ hybridization.
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 http://cbio.mskcc.org/ approximately aarvey/repeatmap.
Arvey
Aaron
A
Computational and Systems Biology Center, Memorial Sloan-Kettering Cancer Center, New York, NY 10065, USA.
Hermann
Anita
A
Hsia
Cheryl C
CC
Ie
Eugene
E
Freund
Yoav
Y
McGinnis
William
W
D000818:Animals; D029721:Drosophila Proteins / Q000235:genetics; D004331:Drosophila melanogaster / Q000196:embryology / Q000235:genetics; D004625:Embryo, Nonmammalian / Q000737:chemistry; D017404:In Situ Hybridization, Fluorescence / Q000379:methods*; D009687:Nuclear Proteins / Q000235:genetics; D015347:RNA Probes / Q000737:chemistry*; D012333:RNA, Messenger / Q000032:analysis* / Q000737:chemistry; D012091:Repetitive Sequences, Nucleic Acid; D014157:Transcription Factors / Q000235:genetics
D029721:Drosophila Proteins; D009687:Nuclear Proteins; D015347:RNA Probes; D012333:RNA, Messenger; C059627:Scr protein, Drosophila; D014157:Transcription Factors; C066591:abd-A protein, Drosophila