Genetic Engineering & Biotechnology News

DEC 2018

Genetic Engineering & Biotechnology News (GEN) is the world's most widely read biotech publication. It provides the R&D community with critical information on the tools, technologies, and trends that drive the biotech industry.

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GENengnews.com | Genetic Engineering & Biotechnology News | DECEMBER 2018 | 15 GEN: How are miRNA research tools and reagents being improved? Dr. Horejsh: Promega will soon launch a Maxwell ® puri- fication kit for miRNA from plasma, serum, and exosomes. The Maxwell RSC is a small, automated instrument for 1–16 samples that performs many of the steps in the purifica- tion. The instrument purifies miRNA from a preprocessed sample without the need to perform manual binding, washing, and elu- tion steps. The chemistry is an extension of our Maxwell RSC miRNA Tissue Kit that has changed the way that our collaborators are able to study miRNA expression. We are also looking at the possibility of moving this workflow to large automation. Dr. Angeloni: Because of the diversity of miRNA ex- pression patterns that can be associated with several cancerous and noncancerous dis- eases, the ability of technologies to detect different patterns of miRNA expression in samples of limited size is becoming more im- portant. In addition, as with other diseases where expression patterns of markers may vary between individuals, the ability to ana- lyze expression pattern differences between different individuals, for research and diag- nostics purposes, requires the use of new as- say technologies and bioinformatics analysis tools. In both areas, multiplexing assay plat- forms and new data analysis tools are pro- viding solutions to these challenges. GEN: MicroRNAs recently have been employed diagnostically using liquid biopsies. Are there any exciting other new applications for miRNAs on the horizon? Dr. Horejsh: I believe that we are still early in our under- standing of miRNA biology. Much as we've seen growing interest and utility of circu- lating cell-free DNA [cfDNA] in oncology research, miRNA will continue to grow in importance. I look forward to better under- standing how exosomes, proteins, circulat- ing cfDNA, and miRNA can be used to more universally study cancer early in disease course to tip the balance toward remission or cure over progression. Dr. Angeloni: With changes in miRNA expression or se- quence variations contributing to the de- velopment of different diseases, this opens the door for new therapeutic targets. As a result, the use of miRNAs as therapeutic agents has been tested with some success. Another exciting approach is the develop- ment of miRNA-targeting drugs. Several companies are developing drugs targeting miRNAs for cardiovascular diseases, some infectious diseases, metabolic diseases, can- cer, and some other noncancerous diseases. Because miRNAs are involved with a broad range of human diseases, the development of new effective miRNA targeted treatment strategies has a bright future. Circulating MicroRNAs as Biomarkers A challenging but potentially rewarding application of miRNA profiling technol- ogy is to identify circulating biomarkers. Such biomarkers could be sought in patient samples obtained by noninvasive means, permitting repeat sampling and disease and treatment monitoring. As Dr. Tewari and colleagues noted in their review, however, "there is increasing concern around the need for rigorous control of preanalytic and ana- lytic variables when considering potential circulating miRNA markers." This concern was mentioned in the review back in 2012, and it still p e r t a i n s . To a d - dress this concern, Dr. Tewari recently led an effort to pro- mote the reproducibility of liquid biopsy studies that quantitate miRNA by means of RNA sequencing. "Different people are using different meth- ods to sequence small RNA, and sometimes getting different results," noted Dr. Tewari. "If it keeps going on like that, it will be hard for the field to make progress." As described in Nature Biotechnology, Dr. Tewari's team prepared samples identically and sent them across the country for each of nine labs to analyze. Each lab used multiple testing protocols to sequence four different samples, including a plasma sample and three synthetic RNA samples. Altogether, the labs tested nine different sequencing protocols, in- cluding four commercially available kits and five protocols developed by the labs. "Both protocol- and sequence-specific biases were identified, including biases that reduced the ability of small RNA-seq to ac- curately measure adenosine-to-inosine edit- ing in miRNAs," the article's authors wrote. "We found that these biases were mitigated by library preparation methods that incor- porate adapters with degenerate bases." "We realized," said lead study author Ma- ria D. Giraldez, M.D., Ph.D., a postdoctoral research fellow in Dr. Tewari's lab, "that not only different methods produce different re- sults, but also any small change within a giv- en protocol can introduce an important de- gree of variation. To compare results across labs, it is key to use a common and highly standardized protocol." According to the study, different se- quencing methods produced different and often inaccurate abundance estimates for individual markers. More encouragingly, the study also indicated that these estimates could be improved by using the methods developed by the consortium labs. Finally, as a University of Michigan press release noted, when RNA sequencing was used to compare the relative amounts of individual miRNAs between different samples, all the methods produced accurate and reproduc- ible estimates. Developers of the original Pipet-Aid ® Pipetting, Pipetting, Pipetting. ...Our Reputation is Built on Reliability If you're tired of unreliable equipment, visit drummondsci.com because DRUMMOND means QUALITY. Go to ergonomic.expert to see how the superior ergonomic design of the Pipet-Aid ® XL can change the way you think about lab hood pipetting. 0ARKWAY"OXs"ROOMALL0!s miRNA Proling Continued from page 13 OMICS Stephen Angeloni, Ph.D. Senior Field Applications Scientist, Luminex MicroRNA is derived from pre-miRNA, which folds back on itself to form a stem- loop structure containing a small region of double-stranded RNA. The loop is removed by Dicer, leaving two complementary RNA molecules—a passenger strand, which is subject to degradation, and a guide strand, which is integrated into the RNA-induced silencing complex (RISC). The guide strand, or miRNA, may attach to complementary mRNA, in which case RISC's Argonaut protein degrades the mRNA. The miRNA may also bind to nearly complementary mRNA, inhibiting translation. Because it needn't rely on perfect complementarity, one miRNA can silence hundreds of genes. National Institute of General Medical Sciences

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