New approach to RNA sequencing reveals thousands of unidentified RNAs in blood

New approach to RNA sequencing reveals thousands of unidentified RNAs in blood

Advancing technology is allowing scientists increasingly to search for tiny signs of cancer and other health issues in samples of patients’ blood and urine. These “liquid biopsies” are less invasive than a traditional biopsy, and can provide information about what’s happening throughout the body instead of just at a single site.

Now researchers have developed a new method for lifting the genetic fingerprints of tiny fragments of RNA found in blood plasma that are invisible to traditional methods of RNA sequencing.

These messenger RNAs and long non-coding RNAs can provide important clues about the activity of genes throughout the body — including genes that are active in particular organs or that are associated with certain diseases, like cancer — and thus could serve as potential biomarkers for a host of conditions.
"The real innovation here 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,"said the senior author, who published the results in The EMBO Journal.

The new method, called phospho-RNA-seq because of the way the fragment ends are tailored, 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.

Authors report that plasma contains fragmented mRNAs and lncRNAs that are missed by standard small RNA-seq protocols due to lack of 50 phosphate or presence of 30 phosphate. These fragments were revealed using a modified protocol (“phospho-RNAseq”) incorporating RNA treatment with T4-polynucleotide kinase, which were compared with standard small RNA-seq for sequencing synthetic RNAs with varied 50 and 30 ends, as well as human plasma exRNA.

Analyzing phospho-RNA-seq data using a custom, high-stringency bioinformatic pipeline, authors identified mRNA/lncRNA transcriptome fingerprints in plasma, including tissue-specific gene sets.

In a longitudinal study of hematopoietic stem cell transplant patients, bone marrow- and liver-enriched exRNA genes were tracked with bone marrow recovery and liver injury, respectively, providing proof-of-concept validation as a biomarker approach.

“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,” the senior author says.

Phospho-RNA-seq has potential applications for discovery sciences as well as more direct applications, the senior author notes.

 “I look at this as proof-of-concept research, but I expect that as we continue to refine the technology and make it even more accessible for other researchers, it’s likely to be applied to many different disease areas and body systems,” the senior author adds.