RNA FISH: a primer
For a long time we’ve been able to pinpoint the subcellular location of proteins, and the advent of FISH (Fluorescence in situ Hybridization) allowed us to locate the position of genes in the nucleus, but recent advances in RNA FISH are making it easier and easier to collect the same data about individual mesnger RNAs.
The Mechanism Behind FISH
For the uninitiated, fluorescence in situ hybridization is a method where specific DNA or RNA quences are visualized inside a fixed and permeablized cell by annealing a labeled nucleic acid probe to the quence of interest. Unlike proteins, where antibodies specific to the endogenous protein of interest have to be raid v ia random massive parallel screening in a biological system (in other words, squirt it in a bunny, mou, goat, or undergraduate*, and screen the rum), we already have a method to generate a specific probe to a known quence – synthesize the complementary quence. This method was originally, and is most popularly, ud against DNA targets. Given a sufficiently specific probe and appropriately stringent annealing and washing conditions, the FISH probe is much like a magnet ud to find a needle in a real haystack: it is attracted to and binds only to its complementary quence in the complex genomic haystack.
Standard RNA FISH
It wasn’t long before the methods that were developed for DNA FISH were applied to RNA and they worked (assuming you skipped the RNa step in the DNA FISH protocol). Some of the first RNA FISH experiments actually ud labeled antin RNA as a probe, however most labs switched over to using DNA probes developed for DNA FISH. There are veral different approaches ud to generate the labeled DNA probes ud in FISH, but the two most popular methods are PCR and Nick Translation. In the PCR method, the probe is simply generated by PCR of the quence of interest, doping the reaction with a dNTP covalently attached to a label, and incorporated into the DNA by the thermostabile polymera. The Nick Translation method modifies a purified plasmid or DNA fragment containing the quence of interest by replacing some of the bas in the DNA with covalently labeled bas, using a ries of enzymatic steps. The two methods are the most popular, as they incorporate many labels per probe, increasing the signal to noi
ratio of the data, and produce long probes, allowing very stringent annealing and washing conditions which limits fal positive signals.
sufferThe DNA probes can be labeled using two different approaches. The first is direct attachment of fl
uorophores, while the cond is attaching a non-fluorescent molecule that will bind strongly to a labeled protein post-hybridization, most commonly using the
biotin/streptavidin system. While direct attachment of the fluorophore is the most straight-forward, t he u of a biotin tag followed by incubation with labeled streptavidin allows for signal amplification,as each streptavidin (or other labeled avidin) will likely contain veral labels. Similar systems might utilize a covalently incorporated antigen with a labeled antibody, such as digoxigenin and anti-digoxigenin, but the concept of signal amplification remains the same.
Figure 1. By labeling the probe with a small molecule like biotin and applying a labeled binding protein like streptavidin, much greater signal intensity can be achieved compared to direct labeling of the probe with fluorophore.
While this signal amplification increas the nsitivity of the method, it increas the complexity of and number of variables in an experiment. In addition, it can come at the cost of increasing background signal if the protein reagents bind/stick to other features in the sample. This isn’t to be trivialized – changes in background signal can vary widely with the source of the reagents, the lot of the reagents within one supplier, cell line, fixation and dehydration method, academic age of the scie
postmasterntist, pha of the moon, and state of the economy, among other things. In addition, the background issues have to be parated from the variables already involved in the hybridization of the probe.
Despite the issues, the robustness of the system and signal made enzymatically generated probes the preferred method for RNA (and DNA) FISH, with background issues worked out through trial and error, and technique knowledge retained by the rich training traditions of academic labs. (“No, you spin to the left after washes. You only spin to the right after you add the probe. Now the whole thing is ruined…”) Using the approache s, rearchers have been able to e localization of RNA from transcription to maturation, and have been able to visualize incread transcription of particular genes as an increa in fluorescent signal in respon to stimuli.
The Next Step In RNA FISH
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月光传说However, there remained one question that the methods described so far could not answer: How many mRNAs of a particular species are prent in a particular cell? This becomes complicated in FISH systems using methods that incorporate a variable number of labels per probe in addition to using condary signal amplification system that has a variable number of fluorophores per streptavi
din. This results in the amount of fluorescent signal generated per probe binding event being highly variable. Put simply, some spots are
brighter than others. So now if you have a bright spot, is it one bright probe bound to one mRNA, or two or three mRNAs bound by probes of average brightness? The desire to accurately quantitate the number of RNAs in each cell has driven rearchers to investigate the u o f chemically-synthesized, labeled oligonucleotide probes for RNA FISH.
While oligonucleotide-bad probes have defined signal per probe, the probes have two major drawbacks: very low signal per probe and potentially reduced specificity compared to the longer probes. The low signal is easily overcome by simply using a panel of oligos that all anneal to the same target, since they each have a relatively small footprint on the target. This approach yielded some excellent results for the Kevin Fogarty and Robert Singer labs when the rearchers ud a handful of multiply labeled oligos per target, but the method was slow to catch on due to the difficulties (and perhaps the ex pen) of synthesizing and purifying oligos that contain veral internal labels.小学生免费学习网
The breakthrough was to think beyond a handful of multiply labeled probes and extend the idea out t
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o 30-48 (or more) independent, singly-labeled oligonucleotide probes per RNA species. This number of probes not only increas signal by putting more fluorescent labels on the RNA, but also becau the probes bind cooperatively. Although RNA is often drawn as a linear, extended molecule, in reality it is a highly folded structure, ba pairing with itlf both locally and distally to form very stable condary and tertiary structures. This folded structure is very difficult for a short DNA oligo to bind to, and this likely frustrated earlier attempts to u oligos as probes. However, once one oligo does bind, it partially destabilizes that ction of the RNA fold, allowing the next oligo to bind more easily, and the next oligo even more easily, and so on, denaturing the structure of the RNA.
Figure 2. RNA (black) can adopt a highly stable folded structure that resists hybridization with small DNA probes (red). However, with a large number of different probes complementary to the RNA, annealing becomes cooperative, resulting in well labeled RNA.
The specificity issues are a little more difficult to deal with, but can be addresd by excluding probe
s that bind to regions of the RNA that are of low complexity (ie. quence regions that aren’t very discriminating to your gene of interest) and matching the annealing energies (Tm) of the various oligos ud. In addition, off target interactions between one particular oligo probe and a RNA that shares some homology with the RNA of interest likely wouldn’t benefit from the cooperative binding described above.
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英语改革This method was shown to be effective in a Nature Methods paper published out of Alexander van Oudenaarden’s and Sanjay Tyagi’s labs at MIT and Ne w Jery Medical School, respectively. In this and subquent papers, it has been shown that this method can robustly detect single mRNA transcripts, and produces some simply stunning images. The method can be applied to both cells and tissue in a variety of species, and has been demonstrated in human, mou, rat, yeast, Drosophila, and C. elegans.
Figure 3. Spectacular examples of RNA-FISH in action, kindly supplied
by Bioarch Technologies. A: GAPDH mRNA (red) + GAPDH protein (green) in HEL-299 cells, B: Over-expresd EGFP protein (red) + GAPDH mRNA (green) in HEK-293 cells
积极的生活态度To make things even simpler, there are now companies streamlin ing the whole process. They take t
he quence of the RNA that you’re interested in, design a suite of matched oligonucleotide probes side-stepping any dodgy areas of the quence likely to cau background, and then synthesize them for you, labeled with your choice of fluorophore. Have any hints or tips for RNA FISH (or DNA FISH, for that matter)? We would love hear about them in the comments!hotshield
