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Sample to Insight Daniel Enderle 1 , Alexandra Spiel 1 , Romy Mueller 1 , Charlotte Romain 2 , Emily Berghoff 2 , Susan Belzer 2 , Stefan Bentink 1 , Martin Schlumpberger 3 , Markus Sprenger-Haussels 3 , Jonathan Shaffer 4 , Eric Lader 4 , Johan Skog 2 , Mikkel Noerholm 1 1 Exosome Diagnostics GmbH, Martinsried, Germany. 2 Exosome Diagnostics Inc., Cambridge, MA, USA. 3 QIAGEN GmbH, Hilden, Germany. 4 QIAGEN, Maryland, USA. Conclusions • The exoRNeasy Serum/Plasma Maxi Kit can be used to extract high-quality RNA from plasma EVs using a fast and convenient spin-column procedure. • EVs contain small and large non-degraded mRNAs. • EVs contain plasma mRNA and a specific fraction of miRNA. • EVs enable detection of somatic mutations and oncogene expression. • Sensitivity of oncogenic transcript detection is highly dependent on sufficient sample volume This work was supported partly by the m4 spitzencluster initiative. The applications presented here are for molecular biology applications. They are not intended for the diagnosis, prevention or treatment of a disease. For up-to-date licensing information and product-specific disclaimers, see the respective QIAGEN kit handbook or user manual. QIAGEN kit handbooks and user manuals are available at www.qiagen.com or can be requested from QIAGEN Technical Services or your local distributor. Trademarks: QIAGEN ® , (QIAGEN Group), Illumina ® , MiSeq ® (Illumina, Inc). Registered names, trademarks, etc. used in this document, even when not specifically marked as such, are not to be considered unprotected by law. © 2014 QIAGEN, all rights reserved. Introduction Extracellular vesicles (EVs or “vesicles”) are a rich source of high-quality RNA, released by all virtually all cells and present in all biofluids. Many current protocols to isolate vesicles use ultracentrifugation; however, development of faster, more convenient methods with high specificity for vesicles and good RNA quality is an area of active research. Here we present a characterization of vesicle RNA isolated from plasma and serum by several different methods, including ultracentrifugation and a novel spin-column-based method. The RNA was subjected to electrophoresis, RT-qPCR arrays and NextGen Sequencing to demonstrate the utility of vesicle RNA as a source for blood-based biomarkers. Figure 1. The new exoRNeasy Serum/Plasma Maxi Kit. Disposable spin column to purify vesicles and workflow of the exoRNeasy Serum/Plasma Maxi Kit. ■ ■ Bind vesicles to membrane and wash ■ ■ QIAzol lysis and release of RNA ■ ■ Phenol/chloroform extraction ■ ■ Ethanol conditioning ■ ■ Bind RNA to RNeasy MinElute ® column ■ ■ Elute RNA 100 bp 4 kb Cancer sample 2 ml plasma exoRNeasy Ultracentrifugation Figure 2. exoRNeasy and ultracentrifugation give similar RNA yields from vesicles present in blood plasma. Bioanalyzer sizing of vesicle-derived RNA purified by two methods. The plasma was pre-filtered (0.8 µm) to exclude larger particles and subjected to either ultracentrifugation, the current gold standard of vesicle isolation, or the exoRNeasy procedure. Both methods purify RNA of similar size and yield. exoRNeasy total RNA 25 200 1000 4000 [nt] [FU] 30 20 10 0 Small RNA fraction Large RNA fraction 25 200 1000 4000 [nt] [FU] 20 0 Figure 3. EVs contain both, small and large RNAs. RNA from 2 ml of pre-filtered plasma was separated into a large and small fraction using different ethanol con- centrations for binding to the RNeasy purification column. Subsequently, both fractions where analyzed using a Bioanalyzer Pico assay. The presence of sharp ribosomal RNA peaks demonstrates the purification of large, intact, non-degraded RNAs from EVs. Small and large RNAs in extracellular vesicles N/A* Kit B Kit S Kit I Ultra exoRNeasy Ultra exoRNeasy Individual 1 Individual 2 Individual 3 Individual 4 Individual 5 Plasma Serum *No procedure available for processing of high volumes 25 27 29 31 33 35 C T Value Figure 7. exoRNeasy enables extraction from high volumes of plasma and serum. Vesicles from 4 ml of plasma or serum were isolated with exoRNeasy, ultracentrifugation (Ultra) and three commercially available methods based on filtration (Kit B) or precipitation (Kit S, Kit I). Only exoRNeasy and ultracentrifugation efficiently recover RNA from high sample volumes. 10% KRAS G12D 25,000 5000 No. of reads WT MUT No. of reads single variants wild type KRAS 100% 20% Frequency Figure 8. NextGen Sequencing detects somatic mutations from CRC in vesicle RNA. A 2 ml sample of pre-filtered plasma was drawn from a patient with KRAS G12D positive colorectal cancer (CRC). RNA from vesicles was isolated using exoRNeasy and analyzed with targeted re-sequencing on an Illumina ® MiSeq ® . Over 10% of all reads that matched to the KRAS gene carry the c.35 G>A/p.G12D mutation previously identified in the primary tumor. RNA recovery with different methods and detection of oncogenic mutations 3 kb from polyA EGFR 1 kb from polyA BRAF 5 kb from polyA KRAS 1 kb from polyA HPRT1 1 kb from polyA GAPDH 28 30 32 34 36 38 40 42 28 30 32 34 36 38 40 C T value RT with random hexamers C T value Reverse transcribed with oligo dTs 42 Figure 4. Extracellular vesicles contain full-length mRNAs with intact poly A tails. RNA from 2 ml of pre-filtered plasma was extracted with exoRNeasy and subjected to reverse transcription with the superscript III enzyme using either random hexamers or oligo-dTs and 6 replicate reactions for each. Both methods yield comparable C T s in RT-qPCR, demonstrating that the quantified mRNA transcripts are not degraded. Input volume, ml C T value (90 mRNAs, pre-amplified) 46% 30 35 40 25 20 15 0.2 1 2 4 Detected Not detected Figure 5. mRNAs from known oncogenes are readily detected in high volumes of plasma. RNA from various volumes of plasma were isolated with exoRNeasy, reverse transcribed and pre-amplified using the RT 2 PCR system and detected using the Human Cancer Pathway Finder PCR Array. At 0.2 ml only 46% of the mRNAs are robustly detected (C T <30) but the isolation with exoRNeasy can be scaled up to 4 ml with linear increase in C T signal and mRNA detection rate. Integrity of mRNA in vesicles and detection of oncogenes in different sample volumes 20 22 24 26 28 30 32 34 36 38 40 exoRNeasy Flow-through 100% capture of mRNA by exoRNeasy vesicle-miRNAs* enriched by exoRNeasy Ago2 miRNAs* in flow-through * Arroyo et al. 2011 PNAS Inside vesicles Outside vesicles BRAF KRAS HPRT1 RPLP0 GAPDH let-7a miR-142-3p miR-92a miR-16 Figure 6. exoRNeasy captures all mRNA and vesicle-specific miRNAs in plasma. RNA from 0.2 ml of pre-filtered plasma was isolated with exoRNeasy and the flow through of the exoEasy column was used in direct lysis. Shown are raw C T values from RT-qPCRs with rows as replicate isolations and colored diamonds as replicate qPCRs. Separation of vesicular from non-vesicular RNA Characterization of RNA from extracellular vesicles
