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Life Tech Expands TaqMan Line with New Assays for Primary miRNAs, Non-coding RNAs

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By Bernadette Toner

Life Technologies' Applied Biosystems group this week launched two new sets of TaqMan real-time PCR assays targeted at the rapidly expanding market for RNA research.

The products comprise TaqMan Pri-miRNA Assays, which are designed to measure the activity of primary microRNAs; and TaqMan Non-coding RNA assays, designed to measure the expression of long non-coding transcripts.

The products, which join a line of TaqMan assays for mature microRNAs that Applied Biosystems initially launched in 2005, are intended to solidify the company's position in the highly competitive RNA research market.

Iain Russell, senior product manager for genomic assays at Life Technologies, told PCR Insider that the company has seen "tremendous growth" for its existing miRNA assays, "and we don't see that slowing, at least for this year."

He said the company expects to see "similar types of growth in the non-coding RNA field," though he admitted that there are more unanswered questions regarding long non-coding RNAs as opposed to microRNAs.

"I think the research community is still trying to get their hands around what it all means," he said of the non-coding RNA field. "MicroRNA stands out a little bit in that it has a very well defined functionality at this point that is very fundamental to gene expression, but I think … as researchers start to delve into the very large amounts of non-coding RNA that exist then we could see tremendous growth in other classes of non-coding RNA as well."

Pri-miRNA Assays

The TaqMan Pri-miRNA Assays are intended to enable researchers to measure the activity of miRNA genes by quantifying levels of expressed primary microRNAs — long RNA transcripts that contain at least one stem-loop precursor miRNA. These stem-loop precursor miRNA molecules are then processed to generate short, mature miRNAs.

"We've had assays out for quite some time now for mature miRNAs, and those are very well accepted now in the marketplace," Russell said. Now, he added, "customers are interested in understanding expression more at the gene level, so we focused upstream on these initial transcripts that are the direct product of microRNA gene transcriptions."

Russell highlighted several areas where the TaqMan Pri-miRNA assays would be of interest.

In one example, he said, approximately 11 percent of mature microRNAs are expressed from more than one genomic locus. For customers using the TaqMan miRNA assays, "when they see a change in that mature microRNA, it's very difficult to understand whether that's due to a transcriptional change at locus A, locus B, or both loci."

In this case, he said, customers could use the microRNA assays in combination with the pri-miRNA assays "to deconvolute where those changes are occurring."

Another use case would be researchers studying the biogenesis of microRNA, particularly in regard to disease pathways. For that application, pri-miRNA and miRNA assays, "when used in conjunction with each other, enable customers to measure the amount of material upstream to the biogenesis pathway and the amount of material coming out at the end," he said.

The new pri-miRNA assays also address a challenge that customers have had interpreting data from association studies. In some cases, he said, "they get linkage to regions of the chromosome that don’t contain … a traditional canonical gene, or a protein-coding region," so the new assays may help provide additional insight into the association.

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The pri-miRNA assays are based on data in the Wellcome Trust Sanger Institute's miRBase database. There are currently more than 1,200 pri-miRNA assays for human, mouse, and rat.

Russell said that the design process for the assays "posed a little bit of a challenge for us" because the primary miRNA transcripts are not very well characterized.

"There are a certain subset where we know quite well where the transcriptional start and stop exists, and which stem loop structures are in there, and hence which mature microRNAs are transcribed as a result of that particular gene," he said. "However, for many of these we don't have that level of information."

As a result, the Life Technologies team took a bioinformatics approach that involved mapping the stem-loop sequences for the precursor microRNAs in miRBase to the most current version of the human, mouse, and rat genomes.

"In doing so we pick up the flanking genomic sequence" on the genome, Russell said. "We design each assay to be within 500 bases of each stem loop, thereby ensuring that what's being measured is the primary transcript that contains the particular stem loop of interest for the customer."

He said that customers can search for pri-miRNA assays using the name, sequence, or ID for the mature miRNA or the stem-loop precursor. The search results page then enables users to view each assay within the context of the genome. The positions of the pri-miRNA, stem loop, and mature microRNA are displayed together alongside the reference genome so that researchers can see the position of each assay within the primary transcript.

Non-coding RNA Assays

The new TaqMan Non-coding RNA Assays, meantime, are similar to the company's TaqMan Gene Expression Assays, except that they detect only the target non-coding transcript as opposed to one or more coding transcripts of the same gene.

The non-coding assays are based on ncRNA annotations from the National Center for Biotechnology Information and RNAdb. The company is offering more than 14,000 non-coding RNA assays for human, around 10,400 for mouse, and 47 for rat.

Russell said that "a good proportion of the non-coding RNA assays" are also available for content found on the NCode non-coding RNA microarray marketed by the Invitrogen group at Life Technologies.

He said that many researchers looking to get into non-coding RNA research would first carry out a broad profiling experiment with the NCode array. Sets of TaqMan Non-coding RNA Assays could then be used to validate results of profiling experiments.

Future Plans

Russell said that LifeTech's current TaqMan microRNA portfolio includes "assays that quantitate the most downstream molecule and the most upstream molecule," but not the stem-loop precursor miRNA that serves as an intermediary between the two.

"That is something that we're definitely interested in trying to fill out, but we don't have timelines available on that yet," he said.

In addition, he noted that the new assays are currently made to order, but the company would consider "inventorying" them in the future if it sees high enough demand.

Life Tech is also mulling the possibility of offering the new assays on its TaqMan array cards — microfluidic cards that enable customers to run up to 384 assays at one time.

"That would enable customers to set up reactions very rapidly and reduce the amount of pipetting that they have to do," Russell said. "I think there is certainly room for growth in terms of providing additional convenience for the customers around this assay product line."

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