Detection of circulating extracellular mRNAs by modified small-RNA-sequencing analysis

Akat, Kemal M.; Lee, Youngmin A.; Hurley, Arlene; Morozov, Pavel; Max, Klaas E.A.; Brown, Miguel; Bogardus, Kimberly A.; Sopeyin, Anuoluwapo; Hildner, Kai; Diacovo, Thomas G.; Neurath, Markus F.; Borggrefe, Martin; Tuschl, Thomas

doi:10.1172/jci.insight.127317

Cited by 43 publications

(61 citation statements)

References 38 publications

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“…However, we have demonstrated that extracellular ribosomes exist at least transiently in the media of cultured mammalian cells and possibly also in body fluids. In support of the latter, the group of Thomas Tuschl has recently optimized a modified small-RNA sequencing method that permits the identification of mRNA fragments in blood plasma or serum 59 . Strikingly, the authors found that the distribution and length of reads mapping to mRNAs was reminiscent of ribosome profiling, suggesting that the sequenced fragments could be the footprints of ribosomes circulating in biofluids.…”

Section: Discussionmentioning

confidence: 99%

Fragmentation of extracellular ribosomes and tRNAs shapes the extracellular RNAome

Tosar

Segovia

Gámbaro

et al. 2020

Preprint

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A major proportion of extracellular RNAs (exRNAs) do not co-isolate with extracellular vesicles (EVs) and remain in ultracentrifugation supernatants of cell-conditioned medium or mammalian blood serum. However, little is known about exRNAs beyond EVs. We have previously shown that the composition of the nonvesicular exRNA fraction is highly biased toward specific tRNA-derived fragments capable of forming RNase-protecting dimers. To solve the problem of stability in exRNA analysis, we developed RI-SEC-seq: a method based on sequencing the size exclusion chromatography (SEC) fractions of nonvesicular extracellular samples treated with RNase inhibitors (RI). This method revealed dramatic compositional changes in exRNA population when enzymatic RNA degradation was inhibited. We demonstrated the presence of ribosomes and full-length tRNAs in cell-conditioned medium of a variety of mammalian cell lines. Their fragmentation generates some small RNAs that are highly resistant to degradation. The extracellular biogenesis of some of the most abundant exRNAs demonstrates that extracellular abundance is not a reliable input to estimate RNA secretion rates. Finally, we showed that chromatographic fractions containing extracellular ribosomes can be sensed by dendritic cells. Extracellular ribosomes and/or tRNAs could therefore be decoded as damage-associated molecular patterns.

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Section: Discussionmentioning

confidence: 99%

Fragmentation of extracellular ribosomes and tRNAs shapes the extracellular RNAome

Tosar

Segovia

Gámbaro

et al. 2020

Preprint

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“…Extracellular RNA content analysis of human biofluids and extracellular vesicles may provide insights into their biogenesis and reveal biomarkers for health and disease. There are currently four types of sequencing-based total RNA profiling of such challenging clinical samples: 1) the recent modified small RNA sequencing methods 8,9 , 2) the SOLiD total RNA sequening method 12 , 3) the Ion Proton method 13 and 4) TGIRT-sequencing using thermostable group II intron reverse transcriptases 5 . The SMARTer method assessed in our study adds a fifth promising method to the sequencing armory.…”

Section: Discussionmentioning

confidence: 99%

“…Presence of mycoplasma was routinely tested using MycoAlert Mycoplasma Detection Kit (Lonza, Verviers, Belgium). To prepare conditioned medium (CM), 4 x 10 8 MCF-7 GFP-Rab27b cells (20 × 175 cm 2 flasks, 300 ml) were washed once with DMEM, followed by two washing steps with DMEM supplemented with 0.5 % EV-depleted fetal bovine serum (EDS). EDS was obtained after 18 h ultracentrifugation at 100,000 g and 4 °C (SW55 Ti rotor, Beckman Coulter, Fullerton, California, USA), followed by 0.22 µm filtration.…”

Section: Methodsmentioning

confidence: 99%

Performance assessment of total RNA sequencing of human biofluids and extracellular vesicles

Everaert

Helsmoortel

Decock

et al. 2019

Preprint

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RNA profiling has emerged as a powerful tool to investigate the biomarker potential of 18 human biofluids. However, despite enormous interest in extracellular nucleic acids, RNA 19 sequencing methods to quantify the total RNA content outside cells are rare. Here, we evaluate the 20 performance of the SMARTer Stranded Total RNA-Seq method in human platelet-rich plasma, 21 platelet-free plasma, urine, conditioned medium, and extracellular vesicles (EVs) from these 22 biofluids. We found the method to be accurate, precise, compatible with low-input volumes and 23 able to quantify a few thousand genes. We picked up distinct classes of RNA molecules, including 24 mRNA, lncRNA, circRNA, miscRNA and pseudogenes. Notably, the read distribution and gene 25 content drastically differ among biofluids. In conclusion, we are the first to show that the SMARTer 26 method can be used for unbiased unraveling of the complete transcriptome of a wide range of 27 biofluids and their extracellular vesicles. 28 30 31 2 of 15 1. Introduction 33All human biofluids contain a multitude of extracellular nucleic acids, harboring a wealth of 34 information about health and disease status. In addition to established non-invasive prenatal testing 35 of fetal nucleic acids in maternal plasma 1 , liquid biopsies have emerged as a novel powerful tool in 36 the battle against cancer 2 . Although in the past most attention was given to circulating DNA, its more 37 dynamic derivate extracellular RNA may provide additional layers of information. However, RNA 38 sequencing in biofluids is technically challenging. Low input amounts, large dynamic range, and 39 (partial) degradation of RNA hamper straightforward quantification. While sequencing of small 40 RNAs 3 and targeted or capture sequencing of longer RNAs 4 proved to be successful, studies using 41 total RNA sequencing on biofluids are rare. To date, only a few whole transcriptome profiling 42 attempts were made on urine, plasma or extracellular vesicles 5-9 , quantifying both polyadenylated 43 and non-polyadenylated RNA transcripts. However, all these methods suffer from one or more 44 limitations such as short fragment length, low amount of quantified genes or ribosomal RNA 45 contamination. 47The advantages of total RNA sequencing are plentiful. Indeed, detection is not limited to a set of pre-48 defined targets, nor to (3' ends of) polyadenylated RNAs. Next to polyadenylated mRNAs, various 49 other RNA biotypes including circular RNAs, histone RNAs, and a sizable fraction of long non-50 coding RNAs can be distinguished. In addition, the study of posttranscriptional regulation is possible 51 by comparing exonic and intronic reads 10 . Altogether, this generates a much more comprehensive 52 view of the transcriptome. 54Here we aimed to assess the performance of a strand-specific total RNA library preparation 55 method for different types of biofluids and derived extracellular vesicles (EVs). We applied the 56 method on platelet-rich plasma, platelet-free plasma, urine and conditioned medium from...

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“…Extracellular RNAs in human plasma have been avidly pursued as potential biomarkers for cancer and other human diseases (1-7). In healthy individuals, plasma RNAs arise largely by apoptosis or secretion from cells in blood, bone marrow, lymph nodes, and liver, while in cancer and other diseases, plasma RNAs may arise by necrosis or secretion from tumors or other damaged tissues, potentially providing diagnostic information (8)(9)(10)(11)(12)(13)(14). As plasma contains active RNases (15,16), the extracellular RNAs that persist there are thought to be protected from degradation by bound proteins, RNA structure, or encapsulation in extracellular vesicles (EVs).…”

Section: Introductionmentioning

confidence: 99%

“…Although high-throughput RNA-sequencing (RNA-seq) has identified virtually all known RNA biotypes in human plasma, studies aimed at identifying disease biomarkers have focused mostly on plasma mRNAs or miRNAs. mRNAs in plasma and blood have been profiled by RNA-seq methods that enrich for or selectively reverse transcribed poly(A)-containing RNAs (10,17) or by sequencing mRNA fragments with (13,14,18,19) or without (4,20) size selection, and it remains unclear which methods might be optimal for biomarker identification. miRNAs in plasma are analyzed by methods that enrich for small RNAs and neglect pre-miRNAs or longer transcripts (9,19,(21)(22)(23).…”

Section: Introductionmentioning

confidence: 99%

Identification of protein-protected mRNA fragments and structured excised intron RNAs in human plasma by TGIRT-seq peak calling

Yao

Nottingham

et al. 2020

Preprint

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SummaryHuman plasma contains >40,000 different coding and non-coding RNAs that are potential biomarkers for human diseases. Here, we used thermostable group II intron reverse transcriptase sequencing (TGIRT-seq) combined with peak calling to simultaneously profile all RNA biotypes in apheresis-prepared human plasma pooled from healthy individuals. Extending previous TGIRT-seq analysis, we found that human plasma contains largely fragmented mRNAs from >19,000 protein-coding genes, abundant full-length, mature tRNAs and other structured small non-coding RNAs, and less abundant tRNA fragments and mature and pre-miRNAs. Many of the mRNA fragments identified by peak calling correspond to annotated protein-binding sites and/or have stable predicted secondary structures that could afford protection from plasma nucleases. Peak calling also identified novel repeat RNAs, miRNA-sized RNAs, and putatively structured intron RNAs of potential biological, evolutionary, and biomarker significance, including a family of full-length excised introns RNAs, subsets of which correspond to mirtron pre-miRNAs or agotrons.

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Detection of circulating extracellular mRNAs by modified small-RNA-sequencing analysis

Cited by 43 publications

References 38 publications

Fragmentation of extracellular ribosomes and tRNAs shapes the extracellular RNAome

Fragmentation of extracellular ribosomes and tRNAs shapes the extracellular RNAome

Performance assessment of total RNA sequencing of human biofluids and extracellular vesicles

Identification of protein-protected mRNA fragments and structured excised intron RNAs in human plasma by TGIRT-seq peak calling

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