Rna FISH

 

Sample Preparation

Tissue fixation and permeabilization are the two most important aspects of sample preparation for FISH, significantly impacting the quality of the data extracted. Fixation is commonly achieved using 4% paraformaldehyde (PFA), which should be prepared fresh for each usage as PFA in solution forms polymers with long-term storage. An alternative is to use a 1:10 dilution of formalin, which does not need to be prepared fresh each time. The incubation period for fixation is highly variable and dependent on the specific tissue—optimized protocols range from 6h for whole mouse brains to 10min for bacterial cells. This is really where trial-and-error or a literature search are necessary. Similarly, fixation temperature affects the quality of the final signal and requires optimization. Although formaldehyde-based fixation is most common, ethanol-based fixation is also used. This involves the use of ethanol or methanol to dehydrate cells and tissues, and can also be used in combination with formaldehyde.

Permeabilization is usually achieved post-fixation using a detergent such as Tween-20, SDS, or Triton X-100. Concentrations of detergents range from 0.1% to 4% (the bulkier the sample the more detergent used—think whole brain vs sectioned tissue). For applications other than eukaryotic cells a proteinase treatment is often applied as well, especially when the penetration of oligo probes is thought to be an issue. Proteinase K treatment nonspecifically digests RNA-binding proteins, potentially making the target sequences more accessible, but can also destroy the integrity of the tissue when over-incubated.

Probe Hybridization

In this step, RNA-specific probes are applied onto the fixed and permeabilized sample. Since hybridization probes are applied onto the sample in solution and rely on diffusion kinetics to enter the cell, incubation times are relatively long– on the order of hours to days. Additionally, longer molecules move slower, so the longer the probe, the longer the minimum incubation. Specialized hybridization buffers are used to increase the chances of successful probe binding. Most buffers include the following components:

· Formamide: reduces the energy barrier for nucleic acid binding allowing hybridization to occur at lower temperatures
· Vanadyl-ribonucleoside complex: an RNase inhibitor which protects RNA-based probes as well as the target RNA
· Dextran sulfate: a crowding agent that encourages proximity of probes and targets
· Bovine serum albumin (BSA): blocks nonspecific probe binding
· Sheared salmon sperm DNA or E. coli/yeast tRNA: also blocks nonspecific probe binding

The other major variables in the hybridization reaction are salt concentration and hybridization temperature. If you’ve ever done a PCR reaction, you’ll recognize these factors—the same concepts of nucleic acid interaction apply. All of these variables must be optimized for a given probe, although basic starting points are as follows: keep the salt concentration constant (750 mM NaCl, 87.5 mM sodium citrate) and maintain a pH between 7.0 and 8.5, while trying different hybridization temperatures to find the optimal signal with least background/nonspecific binding in a time frame of 12-24hrs. In general, the longer the probe the higher the required hybridization temp.

Washing

Following hybridization, several wash steps are necessary to remove nonspecifically bound probe and remove background signal. In general, washes move from higher to lower salt concentration and lower to higher temperature to force removal of weakly bound probes that cannot withstand the stringency of the washing steps. Additional sample processing can be done at this step as well to improve the quality of the final staining, including quenching autofluorescence with a solution of Sudan Black dye and clearing tissue using organic solvent or commercial reagents use as ClearT. Nuclear staining is also often performed as well prior to FISH signal detection. This is typically done on an epifluorescent or confocal microscope at high magnification.

Probe Design Variables

Probes for RNA-FISH are DNA, RNA, or cDNA strands that range in length from 20 bp to over 1000 bp. The most important criterion is sequence specificity to the target RNA. A typical probe for generalized RNA-FISH is a relatively long (500-1000bp) ssRNA riboprobe produced via in vitro transcription. Riboprobes are usually detected using a fluorophore-conjugated antibody, but DNA probes can also be directly conjugated to a fluorophore. An additional increasingly used probe option is the oligonucleotide probe—actually a set of small DNA oligos that each bind to a specific region of the target RNA. This type of probe offers the highest specificity and is particularly useful for tissue types in which larger probes have difficulty penetrating, as well as for applications that are highly specificity-dependent such as identifying splice variants and single nucleotide polymorphisms (SNPs). Several bioinformatics tools have recently been developed to simplify the process of designing oligo probes, among them ProbeDealer and OligoMiner. Intelligent design of hybridization probes to avoid repetitive elements, nonspecific binding, and cross-hybridization is critical for successful staining.

Controls and Troubleshooting

The last step in the FISH protocol is imaging the final stained sample to (ideally) obtain an image of the target RNA staining pattern. Not as simple as it sounds though– a commonly encountered issue during FISH protocol optimization is uncertainty around whether a positive signal reflects actual RNA distribution (rather than nonspecific probe binding) or whether lack of signal represents true absence of target RNA in the sample or inappropriate staining protocol. Controls are therefore a very important aspect of a well-designed FISH experiment. As in qPCR, a well-expressed constitutive “housekeeping” RNA such as actin can be used as a positive control in order to rule out reagent or method issues. Conversely, a negative control can be generated using reverse complement probes (“sense” probes) which should not be able to bind to the target RNA like normal complementary “antisense” probes. Treatment of the sample with RNase and/or DNase can also rule out nonspecific binding of fluorophore-tagged antibodies since probes should not be able to bind these samples.