Minimizing off-target signals in RNA fluorescent in situ hybridization

Abstract

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/∼aarvey/repeatmap.

Unraveling the association between mRNA expressions and mutant phenotypes in a genome-wide assessment of mice

“…Analysis of the spatial and temporal regulation of genes during embryogenesis is necessary to understand developmental genes. In situ hybridization is a classic technique used to visualize mRNA expression in embryos, although in situ hybridization expression signals are noisy and detection specificity is affected by probe design (31). Changes in gene regulation at earlier stages in embryogenesis tend to result in more severe phenotypes, indicated by a higher frequency of embryonic lethality (32), and enhancers responsible for earlier stages of organogenesis are proposed to be more evolutionarily conserved (33), suggesting that early regulatory activities are especially important.…”

Evaluation of genetic markers for forensic identification of human body fluids

“…The method had been earlier reported by Vandewoestyne et al, (2009) to distinguish male cells in male and female sample mixtures. FISH has also been described in embryo development (Arvey et al, 2010), and molecular cytogenetic studies (Lourov et al, 2005). Williams et al, (2013) there was a significant decrease in peak heights between RNA S-FISH treated and untreated cells in both aged and fresh samples.…”

Detection of ALS3 and EAP1 gene expresssion in Candida albicans and Candida maltosa biofilms by FISH

“…On the other hand EAP1 expression was only identified by FISH expression. In spite of this method let to monitor gene expression when there are enough target to stablish hibrydization and signal detection [65,66,67,68,69,70,71], the viabilility of sessil and planktonic cells might be observed.…”