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International Team IDs Expression Changes, Exons, and Splice Sites in Rat Chronic Pain Model

By a GenomeWeb staff reporter

NEW YORK (GenomeWeb News) – Chronic pain is associated with changes in the expression of thousands of genes in a rat model of the condition, according to a recent online paper in Genome Research.

A team of researchers from the US, Germany, and the Netherlands used messenger RNA sequencing to assess gene expression patterns in rats that had undergone spinal nerve ligation, comparing these rat models of chronic pain with unaffected rats. In the process, the team identified thousands of differentially expressed genes and used bioinformatics tools to map splicing sites and find known and previously undetected exons.

"Using this approach offers greater sensitivity, dynamic range and more efficient, unbiased genetic mapping compared to the previous microarray-based methods and may be an efficient new approach to a wide array of problems in neuroscience research," senior author Andreas Beutler, an oncology and anesthesiology researcher at the Mayo Clinic in Rochester, Minnesota, said in a statement.

Chronic pain not only compromises quality of life for tens of millions of Americans, it also leads to billions of dollars in health costs, lost wages, and other expenses. Even so, relatively little is known about the way genes are altered during the chronic pain process.

Past microarray studies have identified some genes that are differentially expressed in the dorsal root ganglion, peripheral nerves that relay information to the spinal chord, during chronic pain, the researchers noted. But, they added, additional work is needed to get a comprehensive picture of how gene expression shifts during long-term pain processes.

"Conceptually, ultra-high-throughput mRNA sequencing should be capable of overcoming virtually all limitations of microarray technology by permitting de novo capture of the full transcriptome of any experimental tissue," Beutler and his co-authors wrote. Even so, they argued, past sequencing-based transcriptome studies have been plagued by complicated bioinformatics and often rely on databases for mapping splice variants.

Beutler and his colleagues used the Illumina Genome Analyzer II to sequence cDNA libraries from the L4 dorsal root ganglion tissue of eight rats: four rats with spinal nerve ligation of the L5 spinal nerve and four unaffected control rats. Two of the chronic pain model rats and two controls were sacrificed two weeks after the spinal nerve ligation, while two control and two affected rats were sacrificed two months after this procedure.

Two weeks after the spinal nerve ligation, the team detected expression differences for more than 2,000 genes between the chronic pain model rats and control rats. At that time, chronic pain was linked to enhanced expression of 1,289 — or 12.4 percent — of known genes and decreased expression of another 721 — seven percent — of the genes.

Expression patterns shifted slightly after two months, though the researchers still saw increased expression for roughly 14 percent of known genes and decreased expression of about 7.5 percent of known genes in the chronic pain rats. And, they noted, most of the genes that were differentially expressed after two months had also exhibited expression differences at the two-week time point.

Along with their insights into chronic pain-related gene expression, the team used their bioinformatics approach to get a detailed view of mRNAs splicing in the dorsal root ganglion and track down exons that had not been reported previously.

Overall, they turned up 92,628 exons linked to the 10,367 known protein-coding genes as well as another 10,464 previously undetected exons in non-annotated regions of the genome. Among them: 421 sets of exons that apparently represent new genes.

About nine percent of the newly detected exons appear to be up-regulated and nearly 13 percent are down-regulated in the rat pain model, the authors noted.

"Taken together, this study demonstrates how the discovery potential of mRNA-seq can be strengthened by newly developed bioinformatics with a strong statistical foundation," the researchers wrote.

Based on the findings so far, the researchers suggested it may eventually be possible to combat chronic pain or other neurological problems by attempting to reverse some of the transcriptional changes associated with the conditions.

"In the future, neurological diseases such as pain might be investigated as problems of transcriptional reprogramming by integrating complete capture and quantification of the various classes of RNA with genome-wide characterization of the chromatin," the researchers wrote. "Such approaches may ultimately lead to the identification of targets for transcription therapy that might employ molecules exerting an analgesic effect by inducing or suppressing specific gene expression programs."

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