The University of Notre Dame’s Jeffrey Schorey, Ph.D., seeks to harness the power of exosomal RNA as a biomarker for Mycobacterium tuberculosis-infected macrophages. Speaking at the ISEV meeting, Dr. Schorey, professor and associate director, Institute for Global Health, Center for Rare and Neglected Diseases, emphasized the potential advantages of exosomes in tuberculosis (TB) diagnostics—namely, easy isolation from various bodily fluids, potential enrichment of TB components present in bodily fluids, and potential detection of TB RNA, TB proteins, and other TB components from a single source.
“For the sequencing of our cDNA library, which was made from the small RNA isolated from exosomes, we used the Roche 454 Genome Sequencer FLX+ instrument,” Dr. Schorey explained. “Our goal was to define not only the microRNAs present in the exosomes, but other small RNA species. One of the surprising findings from our sequencing results is that the exosomes contained M. tuberculosis RNA.”
From there, “we confirmed that the sequenced RNA was fragments of intact full-length transcripts as determined by RT-PCR of RNA isolated from exosomes released from infected macrophages,” he added.
To date, Dr. Schorey and his colleagues have shown that exosomes produced by M. tuberculosis-infected macrophages have a unique repertoire of miRNAs and mRNAs, and that these microvesicles carry mycobacterial proteins and mRNA transcripts and have immune modulatory activity.
Over at the VU University Medical Center in Amsterdam, immunology Michiel Pegtel, Ph.D. and his colleagues are examining how nonrandom small RNA identified in tumor exosomes show biomarker potential. They are taking a deep sequencing approach for these studies.
“In contrast to closed profiling technologies such as microarray and RT-PCR arrays, deep sequencing has literally opened our eyes on the complexity of the human transcriptome. Our previous work using individual quantitative RT-PCR could demonstrate that human B cells infected with the tumor virus Epstein Barr (EBV) secrete exosomes that contain fully mature and functional virus-encoded miRNAs,” Dr. Pegtel noted.
“At the ISEV meeting, Nobel laureate Dr. Phillip Sharp stressed the importance of quantitation in RNA research. I could not agree more as this applies directly to research concerning transfer of functional RNA via exosomes.”
Dr. Pegtel explained that in his team’s virus model, scientists could show that the transfer of relatively small copy numbers of miRNAs from an EBV-infected B cell into an uninfected recipient dendritic cell could directly influence gene regulation.
“Quantitation of RNA copy numbers in exosomes is important, but perhaps it is less important how many are in one or a thousand exosomes,” he said. “More important is what is actually transferred from one cell to another.”
He and his colleagues are now using the Illumina HiSeq 2000 and 100 paired-end protocol on both cancer cells and secreted exomes. Working in collaboration with the University of Granada’s Michael Hackenberg, Ph.D., assistant professor, Dr. Pegtel et al., have found that, aside from the presence of nearly all small RNA families, many smaller RNA fragments are preferentially sorted or excluded from secretion by exosomes indicating an underlying biology.
“Perhaps more interesting is that these small RNA fragments do not adhere to the general rules for Dicer-dependent miRNA processing,” Dr. Pegtel added. “Thus, deep sequencing has revolutionized not only what we know about RNAs inside cells, but also what is secreted into the extracellular milieu that can be used for diagnostics. Because of the quantitative nature of deep sequencing technology, we have gained additional insights into the potential mechanisms that control secretion of known and newly discovered RNAs via exosomes.”
He said that further research in this area will eventually tell scientists more about cell-cell communication in both health and disease. “Decoding the messages that are sent out by, for instance, cancerous cells will hopefully improve and simplify diagnosis such that we will all benefit from this exciting work,” Dr. Pegtel said.