In head and neck cancer, the cells lining the mouth, nose or throat start growing out of control. One of the causes can be traced to infection with human papilloma virus (HPV), and that is actually good news, because these patients, in whom the cancer is caused by HPV, are more likely to respond well to treatment and recover.

But it’s hard to tell, directly, which cancer resulted from infection with HPV. Clinical labs frequently rely on overexpression of the surrogate marker p16, a human protein that keeps the cell cycle in check, to make the call.

There’s a better way, says Xiao-Jun Ma, chief scientific officer at Advanced Cell Diagnostics (ACD, a Bio-Techne brand). Using ACD’s RNA in situ hybridization (RISH) RNAscope technology to link colored probes to the transcripts of the viral genome itself, scientists could observe viral activity more directly. Very recently, ACD and Leica Biosystems made it easier for clinical labs to adopt these kinds of tests, announcing that Leica’s BOND-III instrument for automated protein staining can now perform the RISH assay, too.

In situ hybridization (ISH) uses short nucleotide probes and dyes to label specific genetic sequences in fixed cells or tissues.

Fluorescence in situ hybridization (FISH) for DNA has a long history in the clinic, where it’s often used to visualize genomic rearrangements common in cancer, or the extra chromosome that causes Down syndrome. Now, RISH too is set to make a difference in the clinical setting. Already, it has enabled research labs to pinpoint the cellular location of coding and noncoding RNAs, probing mechanisms of transcription control and RNA splicing.

FISHing for DNA …

FISH can be used when scientists already know what genome alternation they’re looking for—“a copy number variation, for example, or a translocation,” says Anthony Johnson, CEO and president of Empire Genomics. The company manufactures FISH probes. “FISH can identify these structural variations with a resolution down to five [to] 10 kilobases,” Johnson says. What it typically can’t do is differentiate between smaller aberrations.

FISH probes typically consist of a series of fluorescently labeled DNA strands, each about 200 base pairs long, that bind a complementary stretch of target DNA to generate a fluorescence signal.

Researchers can generate their probes from a variety of sources: plasmids, bacterial artificial chromosomes (BACs), cosmids or synthetic DNA. “BACs are a popular starting material,” says Jack Coleman, director of biochemistry at Enzo Life Sciences. Clinics often purchase the probes, which can cost about $25 per reaction. But scientists interested in saving money, or who are studying gene segments for which no commercial probe exists, can make and label their own probes.

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“Using BACs as starting material has limitations, though,” says Jimmy Jin, associate director of global marketing in cancer genetics at Agilent Technologies Inc. Not all parts of the human genome are available as BAC clones. Secondly, BACs based on natural DNA contain pesky repeats, such as Alu sequences, that are quite common in the human genome. Repeat-rich probes hybridize across the genome, creating a high background signal. “Blocking the sample with DNA that binds those repeats first can help, but this will also dampen the signal scientists want to see,” says Jin. Agilent’s SureFISH probes are made from fully synthetic DNA, eliminating the repeats for a highly specific, low-background signal.

… and RNA, too

By hybridizing specific probes to RNA, whether the coding or noncoding type, scientists can see where it is in the cell.

“If they include probes for introns in unprocessed RNAs, they’ll be able to locate the transcription site,” adds Marcela Soruco, product manager at LGC Biosearch Technologies, which makes probes for RNA ISH. The company’s Stellaris probes consist of a pool of up to 48 synthetic oligonucleotides that bind to their target sequence in tandem.

ACD’s RNAscope probes work a bit differently. The company calls its design “double Z” for the approximate shape its paired probes adopt on an RNA target. Each pair sits down side by side, so the bottom rungs of the two Zs line up with the RNA target. The top rungs contain standard sites that attract multiple fluorescently labeled probes, but only when both Zs are present. In this way, RNAscope is both specific—as paired probe binding is required—and able to amplify the signal from a handful of Z-pairs into a strong fluorescence signal.

“ACD’s newer BaseScope assay is more sensitive, so it can produce signal with just a single pair of Zs [and] detect single base-pair mutations or the individual splice sites produced by alternative processing of a transcript,” Ma says. The assay also could be used to check gene editing achieved with CRISPR/Cas9 or other techniques.

“Automating the RNAscope assay was an important step towards making it clinically useful,” says Ma. For example, RNA ISH could be applied in companion diagnostics that predict if a given drug will help a patient. ACD has deals with Merrimack Pharmaceuticals Inc. and Bayer, which plan to use the technology in companion diagnostics for cancer medications.

Stellaris products are finding use in basic research so far, for example in a recent study of Zika-virus RNA replication [1]. They’re also being used in the plant biology world, where scientists recently used RISH to study the regulation of a genetic locus called flowering locus C (FLC) [2]. The authors labeled the FLC transcripts, which encode proteins that repress flowering, and the antisense transcripts of the same locus, which repress FLC expression and allow blooming. With Stellaris RISH, the researchers could see that transcription only occurs in one direction at a time, and a cloud of antisense transcripts seems to prevent sense-direction transcription.

Co-author Susan Duncan, of the Earlham Institute in the United Kingdom, loves the technique, because it allows her to see exactly where the RNAs are. “It’s very direct,” she says. “Seeing, in this case, actually is believing.”

References

1. Aagaard, KM, et al., “Primary human placental trophoblasts are permissive for Zika virus (ZIKV) replication,” Sci Rep, 7:41389, 2017. [PMID: 28128342] 

2. Rosa, S, et al., “Mutually exclusive sense-antisense transcription at FLC facilitates environmentally induced gene repression,” Nat Commun, 7:13031, 2016. [PMID: 27713408] 

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