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RUCDR, PreAnalytix, NuGen Validate RNA-seq Workflow for Human Whole Blood Samples

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NEW YORK (GenomeWeb) – A partnership between Rutgers University's RUCDR Infinite Biologics, PreAnalytix, and NuGen Technologies has yielded a clinically validated end-to-end laboratory workflow for the collection, storage, transport, and preparation of human blood samples for RNA sequencing.

The new workflow, which was validated across three separate laboratories in New Jersey, is a critical step toward standardizing the use of RNA-seq in clinical genomics by potentially eliminating inter- and intra-laboratory variation, RUCDR Infinite Biologics Chief Operating Officer Andrew Brooks told GenomeWeb this week.

RUCDR Infinite Biologics, PreAnalytix, and NuGen forged their partnership just six months ago in an effort to produce a workflow to prepare whole human blood that would reduce changes to RNA after collection and increase sequencing efficiency.

"We were all very motivated to generate data to be able to guide people as to what to do, because … every day our respective programs get questions about how to do things, how to QC, how much RNA [to use]," Brooks said. "As more people are doing RNA-seq and increasing their sample collections, the number of questions keeps increasing. We were all very dedicated to getting this done quickly."

RUCDR Infinite Biologics is the research services and biorepository arm of the Rutgers University Cell and DNA Repository (RUCDR). It is a service organization that is run out of Rutgers' Human Genetics Institute, but is independent of the school. Currently, RUCDR Infinite Biologics is the federal biorepository for four National Institutes of Health institutes. In addition, the organization has partnered with BioStorage Technologies to create a private sector business called the BioProcessing Solutions Alliance, which uses essentially the same infrastructure to offer these types of services to pharma and biotech customers.

Brooks said that prior to developing the new workflow, preparing whole human blood samples for downstream molecular analyses such as RNA-seq was "a bit of a hodgepodge."

"The typical procedure was really based on recommendations for what our clients and investigators wanted," Brooks said. "And those were based on what sales rep got to them last, or what they read on somebody's website. But the problem was that there was really no clear guidance. And we were really limited by how samples were collected and [analyzed], and not really offering a validated workflow."

The new workflow, which Brooks presented this week at the International Leaders in Biobanking Conference in Toronto, is a marriage of three core technologies. The first is the PAXgene Blood RNA System from PreAnalytix, a joint venture established more than 15 years ago between Qiagen and Becton-Dickinson. The company has offered its PAXgene Blood RNA system since 2001. The system comprises the PAXgene Blood RNA Tube and Blood RNA nucleic acid purification kit, and is intended for the collection, transport, and storage of blood and stabilization of intracellular RNA in a closed tube for subsequent isolation and purification of intracellular RNA.

The partners standardized their extractions using Qiagen's QIAsymphony, a Class I 510(k)-approved instrument, "so for clinical applications there is less validation that has to go on," Brooks noted.

The second piece is NuGen's whole-blood or universal RNA-seq chemistry, which is driven by the company's insert-dependent adapter cleavage (InDA-C) technology. This technology, launched last year, is designed for the effective removal of specific transcripts from RNA-seq libraries without impacting non-targeted transcripts. According to NuGen's website, the technology employs "specific and robust enzymatic steps to eliminate undesirable transcripts such as rRNA, globin, and other housekeeping transcripts during library construction without perturbing the original total RNA population as with hybridization capture methods." The specificity of transcript depletion relies on InDA-C primers, which can be designed to target virtually any class of unwanted transcripts from any species, the company notes.

"You create your whole-transcriptome library, and … based on [InDA-C] you engineer restriction sites or cleavage sites into the unwanted sequences," Brooks explained. "When you treat the library, all of those sites are cleaved and you only PCR amplify the sequences you want. And we showed that it doesn't affect any of the known sequences. It reduces almost to zero the unwanted sequences. If you wanted to do targeted RNA-seq, you could deplete as many sequences as you want, so you enrich through depletion. Integrating that chemistry with the RNA that comes from the PaxGene tubes was outstanding."

The final piece of the workflow is Illumina sequencing chemistry, Brooks said. "We standardized against the NextSeq to maximize the number of reads we can get from RNA-seq in order to get the appropriate coverage for doing not just mRNA analysis and differential gene expression, but also looking for splice variation and structural differences," he said.

After developing the workflow, the partnering organizations validated it across three sites in New Jersey with multiple operators, using several nanograms of total RNA from each of 10 subjects. They found a high degree of reproducibility across sites, operators, and equipment.

The group is currently preparing a manuscript summarizing the data for publication in a peer-reviewed scientific journal. In the meantime, Brooks said that the partners want to disseminate their workflow and data as broadly as possible so that other laboratories can evaluate and potentially adopt the method.

"Instead of just looking at our reads and interpretation, go and interpret the data yourself," Brooks said. "Turn it upside down, inside out, chop it up, and put it back together, so you can convince yourself that the workflow is solid."

The researchers are also providing their study design to clinical researchers that want to do their own independent validation as part of a laboratory-developed test. "We're giving them the blueprint to do the exact same thing," Brooks said. "And we did this based on New York State guidelines so the design would be commensurate with anyone who wanted to get an LDT approved. We're not selling anything, because we're not allowed to do that — the LDT has to be done by the individual lab. But we're just giving people a guideline that we know if they follow it will be accepted."

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