Fluorescent in situ hybridization targeting ribonucleic acid molecules (RNA FISH) is a methodology for detecting and localizing particular RNA molecules in fixed cells. This detection utilizes nucleic acid probes that are complementary to target RNA sequences within the cell. These probes then hybridize to their targets via standard Watson-Crick base pairing, after which one may detect them via fluorescence microscopy, either through direct conjugation of fluorescent molecules to the probe, or through fluorescent signal amplification schemes. Recent advances in RNA FISH have increased the specificity and sensitivity of the method to enable the detection of individual RNA molecules, providing very accurate measurements of of RNA abundance and localization at the single cell or even subcellular level. While most applications thus far have been in fixed cells, advances in probe technology have lead to the ability to detect single RNA molecules in living cells.
The protocols for in situ hybridization (ISH) targeting RNA molecules are well established and conceptually simple. All RNA ISH protocols essentially involve bathing the sample in a high concentration of nucleic acid probe (or probes) that are complementary in some way to the target RNA species, driving hybridization of the probe to the target, a principle derived from the development of DNA ISH (Gall and Pardue, 1969). After hybridization, one washes away excess unbound probe, theoretically leaving only those probes specifically bound to the target molecule. Differences between the variants of RNA ISH typically revolve around the type of nucleic acid used for the probe and the type of labeling scheme used to detect the probe via microscopy. Initially, researchers used radiolabeled cDNA probes complementary to the appropriate target (Harrison et al., 1973). Issues with radiolabeling include the low spatial resolution and difficulties associated with handling and stability of radioactive materials. Thus, the development of fluorescence-based (FISH) approaches using DNA or RNA probes provided a major step forward in the field, first applied to DNA FISH (Bauman et al., 1980) and then RNA FISH (Singer and Ward, 1982). Rapidly, researchers adopted the approach of generating cDNA or RNA probes (via enzymatic amplification or nick translation) containing modified bases (Langer et al., 1981) that allowed the conjugation of various haptens or even fluorophores, thus facilitating either indirect or direct detection via fluorescence microscopy. These probes did present some challenges, however, because it was hard to ensure a consistent and high degree of labeling from experiment to experiment. Moreover, the sensitivity of that style of fluorescent probes is generally poorer than that of radiolabeled probes due to cellular autofluorescence. This is problematic because many important mRNAs (such as those encoding transcription factors) are often present at very low abundances, often on the order of a few molecules per cell or less. Subsequent developments in RNA FISH methodology, however, have resolved many of these problems by refining RNA FISH to the point where it can detect single RNA molecules, enabling the direct quantification of RNA species of very low abundance and providing absolute measurements of RNA copy number.
Broadly, current methods for single molecule RNA FISH fall into two categories: those that use some form of signal amplification, and those that rely on direct detection of signal. Direct detection involves labeling the probes themselves with fluorophores. In order to achieve single molecule sensitivity, the probes must have enough fluorescence to be detectable above background autofluorescence. One technique is to use a set of short single-stranded DNA oligonucleotides complementary to various regions of the target RNA, each labeled with one or more fluorescent moieties (Femino et al., 1998; Raj et al., 2008). The binding of multiple probes localizes enough fluorophores to the target RNA such that the RNA is easily visible as a fluorescent spot via fluorescence microscopy (Fig. 1). The benefit of using several oligonucleotide probes at the same time is that the off-target binding of a single oligonucleotide in the probe pool will either be undetectable or readily distinguishable to the much brighter spots corresponding to the true RNA, thus reducing the chances of false positives. False negatives are similarly unlikely, for even if a single probe out of the pool fails to bind, the rest are likely to bind. Recent research indicates that the use of nucleic acid chemistries with tighter and more specific binding properties such as linked nucleic acids (LNA) may be able to produce similar results using a single probe (Taniguchi et al., 2010), although the detection of just one or few fluorescent molecules may only be feasible in cases like bacteria where cellular autofluorescence is kept to a minimum (as opposed to probe pools, which work in a variety of contexts (Raj et al., 2008)). In general, the signals produced via these methods are low and require the use of sensitive CCD cameras and high NA optics for detection.
In order to circumvent the limitation of low signals from the relatively small numbers of fluorescent molecules targeted to mRNA in these direct detection methods, researchers have also developed a large variety of schemes to amplify signals from individual molecules. Some of these are relatively simple extensions of the direct detection methods, such as detection of the probe by fluorescently labeled antibodies targeting specific haptens incorporated in large numbers into an RNA probe (Paré et al., 2009). Others involve targeting nucleic acid probes with a single or few haptens with antibodies conjugated to enzymes; those enzymes in turn will act upon a substrate in such a manner as to create a fluorescent product that will become covalently linked to surrounding molecules (Kerstens et al., 1995). Yet other methods amplify signals by using DNA polymerase and circular templates to locally create long, repetitive single-stranded DNA tracts in situ, which one then targets with short oligonucleotide probes (Larsson et al., 2010). Such methods have the advantage of labeling targets to such a degree that the signals are easily visible even by eye, precluding the need for expensive optical setups. Also, they are able to reliably detect short RNA molecules such as miRNA (Lu and Tsourkas, 2009), and researchers have even demonstrated the ability to detect single-base differences in RNA molecules (Larsson et al., 2010). However, such methods are somewhat prone to lower detection efficiencies owing to the large number of steps in such protocols, each of which has some probability of failure. Some reports indicate that such issues are relatively minor (Lu and Tsourkas, 2009), while others amplification methods detect only a small fraction of the target mRNA (Larsson et al., 2010). Another point of comparison between direct detection and amplification is the ability to detect different species at the same time by using spectrally distinguishable fluorescent moieties: many amplification methods are limited to a single “color” owing to the necessity of using a single enzyme/substrate pair, whereas direct detection can utilize the plethora of organic dyes with different spectral properties currently available. The adoption of these various methods for performing FISH in fixed cells are likely to depend on the particular demands of the application at hand.
Traditionally, researchers have considered FISH to be a methodology that only applies to fixed cells, as the use of oligonucleotides in living cells has proven to be very challenging for a host of reasons. Chief among these are the fact that cells quickly sequester short single stranded DNA oligonucleotides in the nucleus for rapid degradation, and the inability to wash away unbound probe, leading to high background. A recent study has circumvented these issues by designing a degradation-resistant variant of a “molecular beacon”, which is a short oligonucleotide probe with a fluorophore on one end and a quencher on the other (Tyagi and Kramer, 1996). The probe is designed in such a way that the fluorophore and the quencher are in close proximity when the probe is not bound to the target but are far apart when it is bound to the target; thus, the probe is only fluorescent when bound to the target. This reduces the background to the point where one can detect individual mRNA molecules in living cells (Vargas et al., 2005). Further refinements of these methods may open the door to new applications of these live cell FISH techniques.
RNA FISH has a large number of biological applications, but is particularly useful as an assay for spatial aspects of gene expression. One scenario is one in which there is significant cellular heterogeneity, precluding more traditional bulk biochemical assays such as qRT-PCR or northern blots. For instance, it is commonly used to detect gene expression in the study of developmental biology, where it allows for the detection of gene expression in particular sets of cells within the context of a whole developing organism. It also has applications in fully developed tissue that contains a variety of cellular types that would otherwise be averaged together. It is also useful in the study of subcellular localization of RNAs. Examples include the asymmetries in mRNA distributions in the developing Drosophila melanogaster syncytium (Fowlkes et al., 2008), the localization of ASH1 mRNA to the daughter cell during yeast cell division (Long et al., 1995), and the localization of beta-actin mRNA to the leading edge of cells during cellular migration (Lawrence and Singer, 1986). The development of single molecule RNA detection via FISH allows for the accurate quantification in these contexts, and allows for the detection of the localization of even low abundance mRNAs and non-coding RNAs (Khalil et al., 2009). Single molecule counting also provides absolute quantification, which is also useful even in cases like cell lines in which bulk methods are in common use, providing, for instance, very accurate measurements of variability in gene expression (Raj et al., 2006; Raj and van Oudenaarden, 2009). On the horizon, the development of live single-molecule RNA FISH methods promise to bring a much deeper temporal understanding of transcription and RNA dynamics.
The advent of single molecule sensitivity represents in many ways the ultimate fruition of RNA FISH. Through the establishment of simple, reliable methods, RNA FISH represents in many ways a new gold standard for RNA quantification. On the horizon, the development of live cell versions of FISH promise to animate the already rich three-dimensional picture RNA FISH can yield in fixed cells. With applications across a range of biological fields, RNA FISH is a powerful tool for studying gene expression and RNA biology.
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