Genetic Engineering & Biotechnology News

JUL 2017

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20 | JULY 2017 | GENengnews.com | Genetic Engineering & Biotechnology News See RNA-Seq on page 22 features, scientists at Advanced Cell Diag- nostics developed the RNAscope ® technolo- gy, an in situ hybridization assay that can de- tect target RNA with cell specificity in intact tissues. The RNAscope technology amplifies target-specific signals while simultaneously suppressing nonspecific hybridization that would lead to background noise. "For any candidate genes of interest identi- fied by RNA-seq, we can easily look in intact tissue in context of pathology, to see where that gene is expressed," asserts Christopher Bunker, Ph.D., vice president of business devel- opment, Advanced Cell Diagnostics. "Using an in situ method for RNA detection enables verification of a transcript's cell of origin." "In this assay," he continues, "we use a pool of oligonucleotide probes, which we can design for specific detection of any tran- script of interest, any splice variant, and even mutations identified as being interesting or relevant from RNA sequencing. These are used with a universal set of our RNAscope amplification and detection reagents." According to Dr. Bunker, the method is sensitive, specific, and resolves single RNA molecules within intact cells and tissues. It can be applied to any gene of interest, and it can be multiplexed with cell markers up to 4-plex. "The assay is done on intact tissue," Dr. Bun- ker notes, "but it resembles real-time PCR in terms of its simplicity and sensitivity." One of the applications of the RNAscope assay is to validate findings from single-cell RNA-seq studies. "Wider awareness and adoption of RNAscope in situ hybridization (ISH) as a cross-validation method might re- duce the need to perform single-cell analy- ses," explains Dr. Bunker. "One could very quickly go from identifying any transcript of interest (for instance, a gene that is upregu- lated in disease) to determining which cells in intact tissue are expressing that gene." He adds that because RNAscope and RNA-seq are both capable of delivering re- sults in a very time-efficient manner, they may be performed sequentially. Specifically, the techniques may be used together to examine the targets discovered through sequencing so as to prioritize them. That is, the targets most relevant to disease may be identified. "We have another newly developed RNA ISH assay, a variation called BaseScope," in- forms Dr. Bunker. "It can detect deletion and point mutations in intact tissue." BaseScope is an ISH assay that is based on the same plat- form as the RNAscope technology. According to Dr. Bunker, BaseScope promises value not only for the verifying and validating of mu- tations discovered by RNA-seq, but also for interrogating the heterogeneity of those muta- tions within the tumor tissue. "We have used BaseScope for detection of about a dozen mutations so far, such as KRAS, BRAF, and EGFR," says Dr. Bunker. While RNA-seq can identify those mutations in tumor tissue, it does not necessarily indicate whether they occur homogeneously, through- out the tumor, or whether they represent heterogeneous phenomena. "By doing ISH," insists Dr. Bunker, "it is possible to obtain an actual view of which cells have the mutation versus the wild-type copy." Tracking Temporal Dynamics "We performed a real-time analysis dur- ing the primary T helper (Th)-cell response, tracking changes from RNA polymerase II binding to changes in the transcriptome and translatome," says Elke Glasmacher, Ph.D., group leader, Institute of Diabetes and Obe- sity, Helmholtz Zentrum München. She adds that her team saw this analysis as a way of working toward a display of the "overall comprehensive course of events." It has been known for a long time that the activation of immune cells results in rapid functional changes. Details about the under- lying dynamics of the gene expression events, however, have been more elusive. In a study that combined time courses of 4-thiouridine sequencing, RNA-seq, ri- bosome profiling, and RNA polymerase II chromatin immunoprecipitation sequencing (ChIP-seq), Dr. Glasmacher and colleagues examined the genome-wide temporal dy- namics of transcriptional and post-tran- scriptional changes in approximately 10,000 genes during T-cell activation. This study was the first comprehensive real-time analy- sis of the temporal dynamics of transcrip- tion, splicing, and translation during effector T-cell activation. The analysis revealed that transcription and translation are highly coupled for ~92% of the genes, and it showed that for a few genes, transcription and translation are in- dependently regulated. Gene upregulation mostly occurred by the rapid de novo recruit- ment of RNA Pol II to genetic loci, and the release of paused RNA Pol II from promoter- proximal positions played only a minor role, particularly for immediate-early genes. "Our findings provide a foundation for immunologists to identify previously unno- ticed genes that are highly regulated," de- clares Dr. Glasmacher. These genes, she adds, "might in the end also play a role in certain disease contexts." The study also revealed very different intrinsic translation rates for some of the genes. "For example, it appears that cytokine mRNAs have a different translation rate than other genes," Dr. Glasmacher points out. "We did not analyze this in depth yet, but it will be the foundation for future studies." When transcription and translation rates of activated T cells were compared with the rates found in nonactivated cells, the research- ers discovered there were few differences. However, among activated T cells, intrinsic R The Fragment Analyzer ™ simultaneously quantifies and qualifies RNA samples, whether you're performing total RNA, mRNA or small RNA analysis, or working with degraded materials like formalin fixed tissues. TRANSFORMING RNA ANALYSIS WITH AUTOMATED SAMPLE QC. 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