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Study Indicates RNA Barcoding System Offers Alternative to Microarrays, RT-PCR

NEW YORK (GenomeWeb News) - A new gene expression system that relies on digital detection of color-coded gene transcripts is proving as or more sensitive than microarrays or real-time PCR, according to new research.
Researchers from Seattle-based firm NanoString Technologies, the Institute for Systems Biology, the California Institute of Technology, and elsewhere today reported the results for NanoString’s nCounter gene expression system, which directly measures mRNA expression levels without enzymatic reactions, in the advanced online edition of the journal Nature Biotechnology.
The study, which compared the nCounter platform to the Affymetrix GeneChip platform, Applied Biosystems’ TaqMan PCR, and SYBR Green real-time PCR, found the system to be “more sensitive than microarrays and similar in sensitivity to real-time PCR,” according to the paper.
The nCounter system relies on gene barcodes created using a short capture probe — 35 to 50 bases that anneal to the target mRNA — and a reporter probe — another 35 to 50 bases that anneals to the mRNA and is also linked to a tag marked with a particular fluorophore. Because each transcript has a different tag, they are easily differentiated from one another, even in solution. And the number of times each barcode is detected reflects the gene’s level of expression.
The research team, which also included collaborators from the University of Washington and University College London, profiled the expression of 509 human genes — 347 of which had been characterized in previous studies of poliovirus-infected cells, along with 162 used in previously designed probes. They also looked at an additional 15 control genes. 
By mixing total RNA with probes for the 524 genes in solution, the team hybridized probes to their genes of interest. They then used affinity purification to remove unhybridized probes so that they would not interfere with mRNA detection and digitally measured how many times the code for each mRNA transcript was detected.
Starting with 100 nanograms of total RNA, they reportedly detected gene expression in a reproducible manner. As well, their results suggest the nCounter system is slightly more sensitive than microarray, since it detected 88.4 percent of transcripts as opposed to the 82.6 percent detected using the Affymetrix Human Genome U133 Plus 2.0 microarrays combined with GeneChip Two-Cycle Target labeling. This sensitivity was similar to results by TaqMan real-time PCR.
They also verified their results in additional experiments, evaluating the expression of 21 genes found in the total RNA extracted from developing sea urchin embryos. Through the sea urchin embryos’ development, the nCounter system revealed gene expression patterns similar to those observed with real-time PCR.
“In this paper we show that the [nCounter] technology analyses hundreds of genes in a single reaction, produces measurements that are comparable, or better than, microarrays and are comparable to qPCR,” Roger Bumgarner, director of the University of Washington’s Center for Array Technologies and a co-author on the study, said in a statement.
“[T]he key aspect of this technology is that the method is scalable to large numbers of samples at relatively modest cost. As such, it has the potential to revolutionize the application of gene expression measurements to patient diagnoses,” Bumgarner said.
Consequently, the authors are touting the nCounter technology as a simple but sensitive gene expression system for detecting expression that may otherwise be missed. They suggest that the nCounter system may be particularly useful for high-throughput gene expression experiments, diagnostic fingerprinting, and research into gene regulation.
“The NanoString approach represents one of the first digital RNA counting technologies — and it opens up the possibility of transforming how we think about transcriptomes — especially for transcripts expressed at low levels,” Leroy Hood, president of ISB and chairman of NanoString’s scientific advisory board, said in a statement.
NanoString was spun out of ISB in 2003. The company holds an exclusive license to digital gene expression technology developed in Hood’s lab.

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