Tag Archives: probe design

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