Phospho-RNA-seq Enables Access to mRNA and lncRNA Fragments

Phospho-RNA-seq, a new approach to RNA sequencing reveals thousands of previously inaccessible RNA fragments in blood plasma, according to a paper published today in EMBO Journal by researchers at the University of Michigan Rogel Cancer Center.

"We believe that there are a wide variety of potential clinical applications," says Muneesh Tewari, M.D., Ph.D., senior author of the paper. "For example, in cancer, we're excited about applying this approach to try to detect the earliest signs of autoimmune side-effects from immunotherapies. There's also the potential for early detection of cancer because there are long non-coding RNAs that are fairly specific to certain cancer types."

Tewari has been working on this problem for more than ten year. In 2008, he published a paper describing a breakthrough for detecting microRNAs from tumors in blood plasma. The method's shortcoming, however, was that it wasn't able to detect more prevalent and organ-specific types of RNA, which are often found in fragmentary form.

"The real innovation in this new study was recognizing that these other types of RNA were being missed because they had simple but critical differences that prevented them from showing up in the blood plasma sequencing results," Tewari explains. "We used an enzyme to tailor the ends of these fragments so they would show up in the sequencing. And that relatively simple step revealed that, yes, there are thousands of these additional gene transcripts in the bloodstream." The second critical piece of the puzzle was developing a method for reliably sorting through the flood of sequencing data to filter out false positives and ensure an accurate result.

"We had to figure out how to separate signal from noise—how to remove bits of irrelevant genetic material from bacterial and viral RNAs as well as from our own genome, which add noise to the data," says Ryan Spengler, Ph.D, a post-doctoral fellow who led the data analysis. "When the sequences are really short, they can match to multiple places in the human genome by chance and it's difficult to say which gene they're really coming from."

The new method is called phospho-RNA-seq because of the way the fragment ends are tailored. It was first validated in experiments using a curated pool of RNA—so the scientists knew ahead of time what accurate results should look like. Then, to demonstrate that it could work in a real-world setting, the method was tested on plasma samples collected weekly from two patients who underwent bone marrow transplants at U-M.

"We could track the markers of the reconstitution of their bone marrow after the transplant, as well as changes in the blood plasma RNA that indicated injury to the liver—which lined up with what we knew was happening from their medical records," Tewari says.