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Single molecule RNA-FISH

Our current understanding of lncRNAs expression mostly relies on high-throughput bulk measurements using microarrays technologies or cDNA sequencing. More recently, single cell polymerase chain reaction (PCR) based measurements and single cell RNA-Seq provide the opportunity to explore the population heterogeneity of individual transcripts [208]. These methods, however, still suffer from low sensitivity in targeting low abundance transcripts such as lncRNAs [209]. Single molecule RNA fluorescent in situ hybridization (FISH) provides an alternative in which one can simultaneously obtain single cell quantitative and spatial measurements of a transcript [192].

RNA FISH is an imaging based technique to detect and localize single RNA transcripts in fixed cells based on the Watson-Crick hybridization of complementary nucleic acid labeled probes [210]. First, cells are fixed and permeabilized, then soaked with an excess of labeled probe(s) targeting the transcript of interest, unbound probes are then washed away, and cells are imaged. RNA-FISH protocols mostly differ in the nucleic acid used to generate probes and the type of labeling scheme used to detect the probe via microscopy. The development of florescence based techniques for probe labeling [211, 212] was a significant advancement to the original in situ hybridization (ISH) methods that targeted DNA [213] and RNA [214] using radioactively labeled probes. In traditional FISH, fluorophores are coupled to oligonucleotides by enzymatic means such as nick translation or in-vitro transcription [215] which result in a random distribution of the fluorescent dye along the probe. While eliminating the challenges in the handling and the stability of radioactively labeled probes, traditional FISH methods still suffer from low spatial resolution due to cellular auto fluorescence as well as low consistency of labeling across experiments and do not provide single molecule resolution [192, 210].

Improvements in probe designs, imaging technology and image processing software enabled the development of single molecule RNA-FISH (smRNA-FISH) [216, 217]. The key improvement was the replacement of a single randomly labeled long probe with a set of consistently labeled

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short probes (first with a few 50 pb probes labeled with 3-5 fluorophores [216], and later with 32-48 single label 20bp probes [217]). The binding of multiple probes localizes a sufficient number of fluorophores to the target RNA such that a single RNA molecule is reliably visible as a fluorescent spot via fluorescent microscopy [216, 217]. This provides higher sensitivity as it is unlikely that all of the oligonucleotides target inaccessible regions of the transcript. Using multiple short probes also provide higher specificity since off-target binding of a single oligonucleotide in the probe pool will either be undetectable or clearly distinguishable relative to brighter spots corresponding to the true RNA [217].

Current methods for RNA-FISH can be broadly divided to two main classes: those which measure the signal directly and those which rely on signal amplification. In the direct methods the probe itself is labeled [216, 217]. These direct methods face specificity challenges when the target is too short as well as a limitation of low signal when the total number of targeting fluorophores is small. Later methods try to improve low signal by using more stable oligonucleotides or signal amplification strategies [218-220].

To increase target specificity, a recent approach uses branched DNA (bDNA) technology to enhance sm-FISH [221]. In bDNA smRNA-FISH, signal is detected and amplified only when a pair of consecutive oligonucleotides binds the target of interest thus eliminating the possibility of signal detected from a single or few non-paired oligonucleotides binding to an off-target. In brief, primary pairs of unlabeled oligonucleotides bind the target, a long preamplifier DNA molecule binds such pairs and serves as a trunk to which several fluorescently labeled amplifier probes bind. The bDNA smRNA-FISH multi-step protocol was recently compared [221] to the traditional smRNA-FISH [216] and was shown to significantly enhance the signal-to-noise ratio resulting with clearer signal with similar detection accuracy [221]. While offering significant advancement in automated high-throughput and image analysis in the same levels of accuracy, sm-bDNA FISH still suffers from limited detection of nuclear transcripts (due to low yield in entrance of the long (>100bp) trunk probes to the nucleus) and its current state offers only simultaneous imaging of four different RNA molecules [221].

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Other recent variations of smRNA-FISH use combinatorial labeling of the probe directly to detect multiple transcripts at a time. These include iceFISH which detects up to 20 transcripts at a time and was used to measure gene expression and chromosome structure at once [222] as well as the coupling of smRNA-FISH with optical super-resolution microscopy (SRM) to detect and measure up to 32 genes simultaneously [223]. Another finer resolution approach is SNP-FISH which can detect a specific allele by coupling simultaneous imaging of multiple fluorescent dyes with image analysis of co-localized spots [224]. Taken together, these advances are poising smRNA-FISH to become a high-throughput tool to study gene expression in a single cell with spatial resolution.

RNA-FISH: a tool for understanding lncRNA functions

One area of research that is less explored is the application of smRNA-FISH to detect less abundant transcripts such lncRNAs. Indeed, RNA-FISH was instrumental in detecting the more abundant and well-studied lncRNAs, including XIST, MALAT1, NEAT1, MIAT, and GAS5, uncovering their unique cellular localization patterns and deciphering their interaction with other molecules and its effect on their localization [160, 170, 225, 226]. Offering higher resolution and sensitivity smRNA-FISH was also applied to detect low abundance lncRNAs [62, 158, 195, 196, 227-230] as well as to estimate the population abundance of lncRNAs that are expressed on average at one or less copies per cell (as in HOTTIP [148]).

Besides the detection of individual molecules, sm-FISH can be used to address broader open questions in a systems level. Specifically, if applied to a large and representative set of lncRNAs it can in principle: (1) uncover their cellular localization patterns and reveal common themes, (2) provide an absolute measure to whether lncRNAs are predominantly in the nucleus, an open question that was so far addressed only by relative measurements of RNA-Seq on cellular compartments [118], (3) reveal localization patterns during specific cell states such as mitosis, or in response to perturbation, (4) illuminate on the interaction of lncRNAs with their neighboring gene by detecting them simultaneously.

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Simultaneous analysis of lincRNAs and other molecules may help address the on-going debate of whether lncRNAs that appear to be involved in epigenetic regulation (based on their interaction with chromatin modifiers) are acting in cis or in trans. Indeed, using multi-color smRNA- FISH, perturbations and single cell correlation analysis, lincHox-a1 was recently shown to repress its neighbor Hoxa1 in cis [196]. Moreover, using perturbation and RNA-FISH, Jpx, a neighbor of Xist, was shown to activate Xist expression, surprisingly, in trans [195].

Taken together sm-FISH has a great potential to advance studies of lncRNAs biology, especially in the early steps of investigating a new gene. Yet, it is still not routinely applied to study new lncRNA and a comprehensive survey of the cellular patterns of lncRNAs is still not available.

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