FAQs
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Product Usage:
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Can Stellaris® RNA FISH Probes detect DNA targets?
Stellaris RNA FISH Probes hybridize to RNA. Genomic (double stranded) DNA remains inaccessible as no denaturation step is performed to unwind the DNA. Further, the Tm of the short, single-stranded probes is lower in DNA than in RNA and would therefore be competed away by the complementary strand. Simultaneous RNA and DNA detection using different probe types is not currently supported because the denaturation step needed to unwind DNA will remove the Stellaris RNA FISH probes.
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What types of controls do you recommend for Stellaris® RNA FISH assays?
The type of Stellaris® RNA FISH controls needed is dictated by the experiment set up and the preferences of the researcher. Whereas sense or scrambled sequences are useful in antisense or RNAi silencing, they do not serve as an appropriate negative control in Stellaris RNA FISH. An ideal negative control would be a specific probe set against an RNA that normally is not expressed in the cell/tissue sample to be tested. Alternatively, you may consider using a probe set targeting the RNA from a gene from a different organism and which is absent from your sample, e.g. GFP. Please read this article on how controls can demystify your Stellaris RNA FISH experiment for more details.
Formalin-fixed paraffin embedded (FFPE) tissue may suffer from RNA degradation, sometimes very significantly. In such cases, detection of an RNA from a reference gene can be useful for verification of the experimental technique. One commonly used reference gene is glyceraldehyde 3-phosphate dehydrogenase (GAPDH). We make available several Stellaris ShipReady probe sets that can serve as controls, including human and mouse GAPDH, and DesignReady probe sets for many other organisms.
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Are Stellaris® RNA FISH Probes comprised of DNA, RNA, or non-standard nucleotides?
Stellaris® RNA FISH probes are comprised of DNA oligonucleotides, each singly-labeled with a fluorophore. Their simple structure and small size allow for sample permeabilization without protease treatment. As a result, the Stellaris method is less cumbersome than other FISH methods and more easily integrates with complementary detection techniques, such as immunofluorescence.
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Can Stellaris® RNA FISH probes be used for multiplexing?
Stellaris RNA FISH Probes may be used to detect several targets simultaneously. The number of possible targets that can be detected depends on the available microscope filters that can spectrally separate each dye. Dyes with minimal spectral overlap can be combined as long as they are carefully matched to those filter sets! We recommend verifying the performance of each probe set individually before starting your multiplex experiments. Visit our Stellaris Dyes and Modifications page for specific dye multiplexing recommendations.
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Can I use commercial antifade media with the Stellaris® method?
Our dyes are compatible with many commercial mounting media such as Vectashield® Soft and Hard set, Prolong® Gold, Prolong® Diamond, and Slow Fade® Diamond. We recommend using Vectashield Mounting Medium (Vector Labs, catalog #H1000). Be aware that some commercial mounting media require the sample to cure for an extended period of time. Though this may be convenient for your experimental workflow,we recommend imaging your sample immediately after mounting, as fluorescence will naturally degrade over time. Visit our Stellaris Dyes and Modifications page for more information.
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Can the Stellaris® RNA FISH method be combined with immunofluorescence?
Stellaris RNA FISH can be combined with immunofluorescence (IF), although some antibodies may not be compatible with FISH conditions. The fixation method used will affect the integrity of cellular structures and macromolecular assemblies of RNA and protein. It may also affect the accessibility of the epitope to be interrogated. Three approaches have been successful in combining Stellaris RNA FISH with IF: 1) Just adding in the IF-antibody/antibodies into the hybridization and/or wash buffers. 2) Performing the IF, then re-fixing the sample with formaldehyde, and then performing the RNA FISH. 3) Performing RNA FISH, then re-fixing the sample, and then performing the IF.
The following methods article and supplement outlines a protocol to combine the Stellaris RNA FISH method with immunofluorescence in cells, with details on page 4 of the Supplemental Material:
Imaging Individual mRNA Molecules Using Multiple Singly Labeled Probes. (2008) Raj, A.; van den Bogaard, P.; Rifkin, S.A.; van Oudenaarden, A.; Tyagi, S. Nature Methods 5(10), 877-9.
Another excellent paper combining IF with Stellaris RNA FISH is:
Visualization of Single mRNAs Reveals Temporal Association of Proteins with microRNA Regulated mRNA. (2011) Shih, J.D.; Waks, Z.; Kedersha, N.; Silver, P.A. Nucleic Acids Research 39(17), 7740-9.
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When setting up my Stellaris® RNA FISH assay, why do I have to adhere my cells to a no. 1 coverslip?
For optimum clarity of signal from the individual transcripts, the sample needs to be as close as possible to the lens, and located in a neat uniform plane. The distance to detection includes the thickness of the glass to which the cells are adhered. When using a thicker glass surface, such as a no. 1.5 coverslip, the sensitivity is reduced and distinct Stellaris RNA FISH signals are more difficult to detect.
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Can I use Stellaris® RNA FISH Probes to distinguish RNAs from genes with high homology?
Studies by LGC Biosearch Technologies and our collaborators have shown that RNAs from genes with high homology can be distinguished by Stellaris RNA FISH. Most gene homology resides in the coding region, and if the untranslated regions can be used as targets, then targets with high overall homology can be more easily discriminated. Under the conditions of hybridization in the Stellaris RNA FISH method, oligonucleotides containing two or more mismatches to the target sequence will hybridize only weakly. To identify and eliminate cross-reacting sequences, we recommend applying bioinformatic approaches like BLAST or a multiple sequence alignment.
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Can the Stellaris® method be used for whole mount samples?
The Stellaris method has been successfully employed to examine RNAs in whole-mount zebrafish embryo, C. elegans, and Drosophila as well as in cultured cells, frozen tissue and FFPE (Formalin-fixed Paraffin-embedded) tissue slices 4-20 micrometers in thickness. Visit our Stellaris Citation Center to see all publications citing Stellaris RNA FISH in whole mount applications. Below, you can find examples of publications using Stellaris RNA FISH probes in whole mount samples. If a whole mount sample is of appropriate thickness, then there is potential for the Stellaris RNA FISH technology to perform well. The Stellaris RNA FISH Probes consist of oligonucleotides on average 20 nucleotides in length, which penetrate tissue better than long DNA probes, or assemblies for indirect detection. In thicker tissue sections, i.e. > 20 µm, Stellaris RNA FISH Probes may offer qualitative results sans the ability to effectively count transcripts. Stellaris RNA FISH Probes may not always afford a sufficient signal to visualize transcripts in thick sections.
Oka Y, Sato TN. Whole-mount single molecule FISH method for zebrafish embryo. Scientific Reports. 2015;5:8571. doi:10.1038/srep08571.
Ji N, van Oudenaarden A. Single molecule fluorescent in situ hybridization (smFISH) of C. elegans worms and embryos. Wormbook. 2012;13:1-16 doi: 10.1895/wormbook.1.153.1
Xu H, Sepúlveda LA, Figard L, Sokac AM, Golding I. Combining protein and mRNA quantification to decipher transcriptional regulation. Nature Methods. 2015;12(8):739-42. Doi10.1038/nmeth.3446
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How do I know that the Stellaris® RNA FISH Probe set is binding specifically to RNA and not DNA?
The absence of a denaturation step in the Stellaris® method retains the DNA binding sites within the duplex structure and unavailable for hybridization. Comparisons between assays which include treatment with and without DNase, will reveal any contribution to Stellaris signal from probe set hybridization to DNA.
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Fluorophores and Optics:
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Which fluorescent dye should I choose for my Stellaris® RNA FISH Probe set?
Selection of the fluorescent dye for your Stellaris RNA FISH Probe set should be chosen to best match the specifications of the available band-pass filters sets on the microscope you will be using. Visit our Stellaris Dyes and Modifications page for specific recommendations and considerations for each of our dyes. To learn more about how to align the dye spectra with your filters, check out our blog here (Imaging Stellaris Assays: Get to Know Your Microscope). It is equally important to consider the autofluorescence of the sample types you plan to study. Autofluorescence in tissue samples is more pronounced in the green wavelengths. Fluorescein, as an example, absorbs around 450 nm and emits around 520 nm.. It is easier to discern true signal from autofluorescence if longer wavelength fluorophores, such as Quasar® 570, 670, or CAL Fluor® Red 610 dyes, which emit in the red or far-red, are used instead. Additionally, be mindful that if you plan on using a transgenic cell line that may be expressing a fluorescent protein like GFP or tdTomato, you want to choose a fluorophore with a different fluorescence maximum. We recommend that you use Chroma’s online dye and filter selection software to help you choose.
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Can I use a confocal microscope with Stellaris® RNA FISH probe sets?
We recommend the use of wide-field fluorescence microscopes as an entry point for researchers new to Stellaris and microscopy. However, more advanced microscopists have successfully employed confocal microscopy for imaging their Stellaris RNA FISH assays. Confocal microscopy uses point illumination to limit the focal plane for imaging. While this technique restricts light that is out of focus, it also diminishes the sensitivity of low-light level imaging.
To best detect individual transcript molecules, we encourage the use of conventional wide-field fluorescence microscopes with a 60-100x, 1.3 NA or greater, oil-immersion objective and a cooled CCD camera. The light source should be a mercury or metal-halide lamp (e.g., ExFo Excite, Prior Lumen 200). We recommend starting with a 1 second exposure time. Check out our blog article for tips on imaging Stellaris RNA FISH assays.
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Why is autofluorescence obscuring my Stellaris® RNA FISH signal?
Most cells and tissue types have natural autofluorescence that is more pronounced in the green region of the visible spectrum. It is easier to discern true signal from autofluorescence if longer wavelength fluorophores such as Quasar® 570/670 or CAL Fluor® Red 610 are used. The signal from shorter wavelength dyes, such as fluorescein (emission near 520 nm), is more difficult to detect due to the autofluorescence.
Autofluorescence is generally more evident in tissue samples, is more pronounced in certain cultured cell types, and can have very broad emission that spans the visible spectrum. For example, fluorescence from lipofuscin bodies can be detected from 360 nm to 650 nm. Fluorescence from the lipofuscin bodies is mostly seen as cytoplasmic perinuclear spots which can be distinguished from true single molecules of RNA by being slightly larger, brighter, and by having fluorescence over a broad range of the spectrum. When you’re searching for your RNA of interest in tissue, it is especially important to distinguish the Stellaris RNA FISH signals from autofluorescent features. One way to accomplish this is to collect the light in a secondary unused filter. This will let you confirm that your RNA of interest is only present when you specifically excite the fluorophore you labeled it with. For example, if your probe is labeled with Quasar 670 that means you will excite this dye near 647 nm and it will emit fluorescence at 670 nm. If you try to excite that same molecule at a shorter wavelength with an unused filter, say FITC near 470 nm, you should not see the fluorescence in the same location. If you do see fluorescence using multiple filters or with filters which are incorrect for your dye, then you are likely seeing autofluorescence and not a true RNA molecule. For more information about the prevalence and origin of autofluorescence, please visit the Nikon Microscopy U website.
In addition, the degree of autofluorescence will depend on the fixation method used. Formaldehyde tends to crosslink fluorescent enzyme co-factors, such as flavins, whereas fixation with methanol/acetic acid tends to release them and wash them away from the sample. Prolonged fixation at elevated temperatures tends to exacerbate the autofluorescence. To assess the overall background fluorescence and to determine the contribution of non-specific probe binding, we recommend imaging a second sample in parallel that has not undergone probe hybridization - a no probe control.
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LGC Biosearch Technologies’ Quasar® 670 dye is listed as an alternative to the Cy™ 5 dye. Is it more resistant to photobleaching?
No, both Quasar® 670 and Cy5 share similar photobleaching characteristics. Like the original Cy5 dye, our Quasar 670 is not sulfonated and is based on the same indocarbocyanine as the Cy5 dye giving it nearly identical characteristics. We recommend commercial mounting media such as Vectashield® and imaging longer wavelength dyes first to help preserve the fluorescence signal. Visit our Stellaris Dyes and Modifications page for more information.
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Can LGC Biosearch Technologies' probes (and dyes) be used for fluorescence in situ hybridization (FISH)?
LGC Biosearch Technologies offers Stellaris® RNA FISH probes and associated products for RNA FISH. We do not currently support DNA FISH applications. Historically, FISH probes were cDNA labeled enzymatically with multiple fluorophores. A single synthetic oligonucleotide labeled with a fluorophore is not sufficiently bright for reliable visualization under a microscope and therefore multiple probes are necessary. With Stellaris RNA FISH Probes, multiple probes hybridize in series along the transcript of interest. The combined fluorescence from the probe set hybridized to the transcript can be seen as a diffraction-limited spot in a widefield fluorescence microscope, allowing both the location of the RNA and the copy number per cell to be revealed.
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Product Documentation:
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Where can I find usage information and chemical properties for Biosearch Technologies' products?
Product information sheets contain important usage information and chemical properties. You may download the product information sheets, by clicking on the appropriate link located under the Technical Specs tab of individual product pages. If for any reason you are unable to find what you need on our website, please e-mail our Technical Support team. -
Does LGC Biosearch Technologies make Safety Data Sheets (SDS) available?
Safety Data Sheets (SDS), formerly referred to as Material Safety Data Sheets (MSDS), are available for download under the Technical Specs or Related Info tabs found on most product webpages. If you are unable to access this document or need additional information, please contact our Technical Support team.
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Shipping & Handling:
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How long does it take to manufacture and ship Stellaris® RNA FISH Probe sets?
Turnaround time is 5 to 7 business days for manufacture, with one additional business day for delivery via FedEx Priority. Stellaris RNA FISH probe sets are shipped dry so neither dry ice nor cold packs are required.
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How do freeze thaw cycles affect oligonucleotides?
Oligonucleotides should be subjected to minimum number of freeze-thaw cycles. Therefore, we recommend that you prepare aliquots and store microvials each having sufficient material for a day’s set of experiments and freeze at -20 °C or -80 °C.
“The repetitive freezing/thawing of an oligonucleotide strand can lead to the undesirable decomposition of a sample through the loss of a terminal phosphate group, a base or the entire oligonucleotide unit. This observation relates to the fact that during the freezing/thawing process the oligonucleotide and buffer components isolate from the solvent system. As a result this isolation increases the percentage of similar intermolecular attractions and bond breakages.”
Analysis of the degradation of oligonucleotide strands during the freezing/thawing processes using MALDI-MS. (Analytical Chemistry, Volume 72, Number 20, pages 5092 - 5096, 2000) -
How should orders be placed for shipment to locations outside the United States?
LGC Biosearch Technologies maintains distribution agreements with partners in countries listed on our Distributors webpage. For all other countries, LGC Biosearch will ship internationally subject to local customs restrictions. All payments must be in US dollars.
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I left my oligonucleotides sitting on the lab bench over the weekend. Are they still ok to use?
Dry oligonucleotides are quite stable at room temperature and not likely to degrade over the short term. We recommend they be moved into cold storage upon delivery or as soon as possible. Oligonucleotides in solution at high concentration can be stored refrigerated for a few days or even one week. At lower concentrations, oligonucleotides are more susceptible to degradation. In all situations, Dual-labeled BHQ® probes should be protected from light to avoid photo-bleaching. -
What buffer should I resuspend my oligonucleotide in?
For oligonucleotide suspension, we recommend preparing stock and working solutions using a TE buffer (10 mM Tris, 1 mM EDTA, pH 8.0) made with nuclease-free water. The EDTA serves to protect against microbial contamination. If your experiment cannot tolerate EDTA, you may use 10 mM Tris-Cl buffer. Suspension in water alone should be limited to nuclease-free water at physiological pHs, but is not recommended. Acidic conditions can lead to degradation of the oligonucleotide through depurination.
Notes: Fluorophores are sensitive to photobleaching. To minimize their exposure to light, we recommend using amber microtubes, wrapping the tube in foil, or else placing clear tubes into a box which is impermeable to light. Avoid freeze/thaw cycles by making working solutions and storing them in aliquots at -15 °C or cooler.
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Why were my oligonucleotides shipped at room temperature?
Unless otherwise requested, we will ship our oligonucleotide products in a dried state, at ambient temperature, by overnight delivery. Dried products are very stable at ambient temperature and are not likely to degrade should there be unavoidable delays, e.g. customs clearance. Products in solution are more susceptible to degradation than dried products and must be shipped frozen. -
I received my oligonucleotide order, but the vial looks empty. Where is the oligonucelotide?
Unless otherwise specified, oligonucleotides are shipped in a dried form. It may be difficult to see the oligo at the bottom of the vial as it may be present as a thin film or a miniscule pellet. To ensure that no product is lost upon opening, briefly spin down the vial in a bench top microcentrifuge before removing the cap. Once suspended in buffer, checking the OD at 260 nm will verify the oligo’s presence and concentration.
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