Tag Archives: RNAScope

Don’t eat this FISH-STIC

single molecule FISH (smFISH) is a great way to detect single RNA molecules in fixed cells. The “traditional” FISH uses fluorescently labeled oligonucleotides which directly hybridize with the target RNA sequence. The two most common approaches are the use of 1-5 50-mer oligos, that are labeled with 5 fluorophores, or the use of more than 20 20-mer oligos labeled at one or both ends.
A more recent approach is nicknamed “Christmas tree”, in which 2-3 rounds of hybridizations are done, with only the last round uses fluorescently labeled probed. I’ve expanded on that when I discussed RNAScope.  Other people have independently developed similar protocols (e.g. branched DNA FISH (bDNA), which was recently used to get smFISH transcriptomics in human cells).

Now, a new paper came out with a very similar method whish they call FISH-STIC (Fluorescence In Situ Hybridization with Sequential Tethered and Intertwined ODN Complexes).

However, their images do not compare to the quality shown by the RNAScope or bDNA papers. Maybe its just bad imaging and not the FISH itself, but their images look blurry and doesn’t seem to be up to modern FISH standards. I used 20-mer FISH and can get nicer images.

They also get a few high-intensity spots which the call “mRNA-independent” (since they can also see it outside the cell). To me it means that their protocol was just not optimized – either the washes, or the oligo sequences which should not make complexes like that.

They also seem to have higher background fluorescence compared to RNAScope.

Simultaneous detection of Actb and Actg mRNAs with FISH STIC probes. Source: Sinnamon & Czaplinski (2014) RNA 20:260-266.

Simultaneous detection of Actb and Actg mRNAs with FISH STIC probes. Source: Sinnamon & Czaplinski (2014) RNA 20:260-266.

On the other hand, their beta-actin FISH-STIC looks better than the RNAScope-FISH. beta-actin mRNA is found in 500-2000 copies per cell, so it is not easy to get smFISH. Still, 20-mer FISH that I do looks better than both.

20-mer FISH for beta-actin mRNA in immortalized MEFs. Source: myself.

20-mer FISH for beta-actin mRNA in immortalized MEFs. Source: myself.

Notice also in my image the transcription sites (TS) are clrearly visible, whereas I did not see any TS in their images, which is weird (unless they chose cells without TS on purpose. Don’t know why, since smFISH is a powerful tool to quantify transcription 1, 2).

They did not test FISH-STIC on a less prevalent mRNA, so I cannot comment on how it would look like compared to RNAScope or 20-mer FISH.

Anyway, the methods seem similar, but I think that the RNAScope demonstrates better results than the FISH-STIC. However, I haven’t tried this approach myself. I continue to do FISH with 20-mer probes and so far pleased with my results.

ResearchBlogging.orgSinnamon JR, & Czaplinski K (2014). RNA detection in situ with FISH-STICs. RNA (New York, N.Y.), 20, 260-266 PMID: 24345395
Battich N, Stoeger T, & Pelkmans L (2013). Image-based transcriptomics in thousands of single human cells at single-molecule resolution. Nature methods, 10 (11), 1127-33 PMID: 24097269

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RNAScope – a new FISH in the sea

I recently started a collaboration that involves the use of the RNAScope method. So here’s a short overview of the method.

FISH is a very useful method to observe and quantify specific RNA species in situ. Yet, a major issue is the signal to noise ratio. Single probes can attach non-specifically in the cell and create background fluorescence. One way to overcome this background is to use multiple probes against the same RNA, but at different locations along the RNA. The more probes specifically attach to the RNA, the better is the signal to noise ratio. However, I know from experience that it is still fairly difficult to distinguish true RNA spots from background signal.

RNAScope, developed by a team from Advanced Cell Diagnostics, tries to overcome this problem from another angle. Instead of having multiple labeled probes against the target RNA, they produce two unlabeled tandem probes. These probes contain a short complementary region (18-25 bases), a spacer sequence and a 14-base tail sequence.

Schematic of the RNAscope assay procedure. In step 1, cells or tissues are fixed and permeabilized to allow for target probe access. In step 2, target RNA-specific oligonucleotide probes (Z) are hybridized in pairs (ZZ) to multiple RNA targets. In step 3, multiple signal amplification molecules are hybridized, each recognizing a specific target probe, and each unique label probe is conjugated to a different fluorophore or enzyme. In step 4, signals are detected using a fluorescent microscope. Source: Wang et al. (2012) J. Molec. Diag. 14:22.

Schematic of the RNAscope assay procedure. In step 1, cells or tissues are fixed and permeabilized to allow for target probe access. In step 2, target RNA-specific oligonucleotide probes (Z) are hybridized in pairs (ZZ) to multiple RNA targets. In step 3, multiple signal amplification molecules are hybridized, each recognizing a specific target probe, and each unique label probe is conjugated to a different fluorophore or enzyme. In step 4, signals are detected using a fluorescent microscope. Source: Wang et al. (2012) J. Molec. Diag. 14:22.

After hybridization with the target probes, comes a second hybridization step with a pre-amplifier probe. This is a long probe that contains a complementary sequence to the 28 bases of the two target probes tails (14+14). So, only when the two target tails hybridize one next to the other the pre-amplifier will hybridize. The pre-amplifier contains 20 binding sites for an amplifier probe which in turn contains 20 binding sites for the labeled probe. Thus, for each target probe pair, we get 20×20=400 labeled probes.

That is a large amplification. They suggest having ~20 target probe pairs per RNA. So each RNA is amplified ~8000 fold (assuming 100% efficiency of hybridization) over the background of single labeled probes.

In their paper, they show convincing images of their negative controls (single target probe compared to no-probe). However, they do not supply any statistics (i.e. how many cells/fields they observed, how many biological repeats, are there cells with some detectable spots?)

Validation of RNAscope. HeLa cells were hybridized with either the full set of probes to 18S rRNA, the left half of the set, or the right half of the set (as shown in the schematic along the top). A no-probe control was performed in parallel as an indicator of background staining. Cells were counterstained with DAPI (blue), which masks nucleolar 18S RNA. Source: Wang et al. (2012) J. Molec. Diag. 14:22.

Validation of RNAscope. HeLa cells were hybridized with either the full set of probes to 18S rRNA, the left half of the set, or the right half of the set (as shown in the schematic along the top). A no-probe control was performed in parallel as an indicator of background staining. Cells were counterstained with DAPI (blue), which masks nucleolar 18S RNA. Source: Wang et al. (2012) J. Molec. Diag. 14:22.

The main object of developing this method, they claim, is to have a good tool for molecular pathology, i.e. – a good method to examine RNA in situ in pathological tissue samples. In their paper, they go on to show RNAScoping of specific mRNAs in cells cultures and in tissue samples. It looks very good.

My associates are going to try this method with my cells within a couple of weeks. We’ll see if it works as good as they claim.

ResearchBlogging.org Wang F, Flanagan J, Su N, Wang LC, Bui S, Nielson A, Wu X, Vo HT, Ma XJ, & Luo Y (2012). RNAscope: a novel in situ RNA analysis platform for formalin-fixed, paraffin-embedded tissues. The Journal of molecular diagnostics : JMD, 14 (1), 22-9 PMID: 22166544