Do you want to directly quantify RNA? Do you want to be able to localize RNA simply, quickly, and precisely? Heard about RNA FISH but are not sure how to go about designing your own probes? Then you want Biosearch Technologies’ DesignReady assays for Stellaris RNA.
We’ve already seen the power of Stellaris® in our previous article RNA Detection with Stellaris Fluorescence in situ hybridization (FISH) probes. Now, you can take the hassle out of designing your own custom probe sets by grabbing a made-to-order DesignReady assay. With Stellaris DesignReady FISH, you can localize and quantify individual RNA molecules in cultured cells, tissue sections, or even whole-mount embryos with a high degree of specificity and sensitivity. Plus, Stellaris DesignReady Assays are easy and affordable.
The Benefits of DesignReady Assays
Rigorous analysis: All DesignReady Assays have undergone bioinformatics assessment to ensure their specificity.
Range of applications: Whether you are assaying apoptosis or studying stem cells, there is likely to be a DesignReady Assay to suit your needs.
Speed: Speed is a big bonus for DesignReady Assays, whether it is the shipping in just 5–7 business days or the fact that you get the results of your experiment the same day you conduct it.
Performing a Stellaris DesignReady FISH experiment
The ease of use of Stellaris DesignReady FISH assays is highlighted by the limited amount of equipment and reagents required:
1. A Fluorescent Scope and Filters
For Stellaris DesignReady you need a widefield fluorescence microscope equipped with a mercury or metal halide lamp. Having the correct filters is also important and depends on your dye of choice. Common dye choices are Quasar 570 dye, CAL Fluor Red 610 dye, and Quasar 670 dye. Note that fluorescein (FAM) is not recommended because of cell and tissue autofluorescence.
Keep in mind that with FISH you will not be able to see most RNA spots with your eyepiece. And while yes, experienced FISH-ers can use confocal microscopy successfully, in general this is not recommended as a starting point, as you run the risk of missing your RNA spots altogether. To learn more about imaging and FISH see Imaging non-coding RNAs with Stellaris FISH Probes.
2. Appropriate Controls
You know the drill: You need both negative and positive controls in your experiments and Stellaris DesignReady FISH is no exception. For positive controls you can use one of Biosearch Technologies’ ShipReady Assays – all of which have been functionally tested and act as a perfect complement to DesignReady Assays. If ShipReady Assay targets do not suit your needs, then you can design your own custom probe set to a gene of medium/high abundance. The point of your positive sample is to make sure your components are working correctly.
For your negative control, a no-probe sample works well. Just remember, other than the absence of probes, to treat this sample the same as all the others. Another, more ideal but not always possible, negative control is to use a cell line or tissue sample that does not express your RNA-of-interest (a knock out). Alternatively, you could use a probe set to an unexpressed gene, such as a reporter gene – but make sure you are confident it isn’t expressed in your sample. These negative controls are necessary to determine the specificity of your signal and identify any auto-fluorescence.
3. High-Quality Reagents
For Stellaris DesignReady FISH you will need some basic reagents: Phosphate buffered saline (PBS), formamide, DAPI, formaldehyde, ethanol, tris-EDTA buffer solution, and vectashield mounting media. Biosearch Technologies’ now offers premade hybridization and wash buffers that can be purchased directly from Biosearch, ensuring you high-quality and consistent results every time. For an example of the performance of these high-quality buffers see Figure 1.
In the lab you often have to make hard decisions about time and money. But, when deciding to make your own buffers or not, remember: Your time is worth money too. And pre-made buffers don’t just save you time in preparation but also valuable time troubleshooting your reagents.
Figure 1: Comparison of Stellaris Buffer with conventional FISH buffers. Panels A-C show Stellaris Buffers. Panels D-F show conventional FISH buffers. The dye used in each case is listed below the panels.
4. Good Probe Design
For FISH you need probes. If Biosearch does not have your gene target of interest, you can use the Stellaris Probe Designer application to custom design your own probes. This application evaluates your target sequence and design probes along with their corresponding position and GC content. The application also analyzes these potential probes for sequences that could hamper their performance (think repeats).
Remember though, probe design by any application is only as good as the sequence you feed into it. Therefore, it is recommended that you test you target RNA sequence by performing BLAST and multiple sequence alignments to determine if it has any similar homologs. Then, exclude those sequences from your design, else you run the risk of cross-hybridization (a lot of background noise).
Also keep in mind during primer design what it is you are trying to detect. For example: If you want to detect mature RNA, design against the coding sequence. If you want to detect multiple variants, i.e. design an inclusive probe set, the input sequence should only be that of the shared sequence. If you want to detect only single transcripts, i.e. design an exclusive probe set, the input sequence should not include common sequences.
Stellaris DesignReady FISH is a straightforward, affordable, sensitive FISH method. Want to learn more? Download the starter guide and read Martha Christie’s article, RNA Detection with Stellaris Fluorescence in situ hybridization (FISH) probes.