Comparison Of Isolation Methods Of Exosomes And Exosomal RNA From Cell Culture Medium And Serum

BACKGROUND&OBJECTIVEExosomes are small particles(30-150nm)secreted by various types of cells by means of endogenous budding and multivesicular body fomation,which enclosed by a phospholipids bilayer and contain DNA?RNA and protein.Exosomes fuction by protecting and transporting biomacromolecule inside them to target cells,they can be released to cell culture medium(CCM),and also found abundant and natural in body fluids,which may be served as biomarkers for development of superior,sensitive and minimally invasive diagnostic alternative in Precision Medicine.However,the extraction methods of exosomes and RNA has not yet been standardized and need to be copared and chosed.The commonly used protocol for exosomes isolation is ultracentrifugation(Ultra),the principle of which is different volume and physical property of vesicles.It conducted by different speed centrifugation,and the final step of which is Ultra at least at 100,000 x g for 70 min to pellet the small vesicles that correspond to exosomes.In addition,ultrafiltration,HPLC-based protocols,immunoaffinity-capture methods single or combined application of Ultra can provide a high enrichment and purity of exosomes.In recent years,easy-to-use precipitation solutions,such as ExoQuick and Total Exosomes Isolation Reagent(TEI)are utilized to isolate and purify exosomes.The novel nanomaterial procedure is convenient and time-saving with no need for expensive equipment or technical challenge.In the downstream analysis of exosomal content,a number of alternative exosomal RNA(exoRNA)extraction methods have been used including phenol based techniques(Trizol),combined phenol and pure column based techniques(miRNeasy,Total Exosomes RNA,Protein Isolation Kit(TEI)and HiPure Liquid RNA/miRNA Kit(HLR)),combined non-phenol and pure column based techniques(SeraMirTM Exosomes RNA Amplification Kit(SeraMir)).The exoRNeasy Serum/Plasma Kits(exoRNeasy)from Qiagen company use a membrane-based affinity binding step to isolate exoRNA directly from serum and plasma.Given highly attractive diagnostic value of exoRNA,available and convenient methods to extract the exoRNA with high quality,substantial yield,purity and appropriate size distribution need to be confirmed.Hitherto,comparative studies of exosomes and exoRNA isolation methods have rarely been conducted.We compared the quantity and quality of exosomes and exoRNA extracted by different methods.Furthermore,in qPCR and high-throughput sequencing analysis,we bring evidence that different combining exosomes and exoRNA isolation methods can impact exoRNA pro filing.We comprehensively evaluated the advantages and disadvantages of different methods and provide the advices of method selection.The study can be divided into three parts as below:Part 1 Comparison of Isolation Methods of Exosomes from Cell Culture Medium and SerumOBJECTIVETo measure and characterize CCM or serum-derived exosomes isolated by Ultracentrifugation(ultra),and two commercially available kits(ExoQuick and TEI)from System Biosciences company and Invitrogen company,and provide suggestions for the choice of extraction methods.METHODS1.To extract exosomes from CCM or serum:CCM or serum after pretreatment were used to extracted by Ultra,ExoQuick and TEI respectively.2.Transmission electron microscope was used to observe the morphological structure and background of exosomes extracted by different methods.3.Bradford method was used to detect the concentration of protein from exosomes extracted by different method;Western Blot was used to evaluate level of the specific protein marker(CD9,CD63 and TSG101)of exosomes extracted by different methods.4.In order to determine the size and quantity of particles isolated by different methods,we used NTA instrument to track each of particles.A video of 60-seconds duration was taken with a frame rate of 30 frames/s,and particle movement was analyzed by NTA software.RESULTSTransmission electron microscopy observations show that exosomes extracted by three methods have similar structure and morphology;Then NTA results were used to compare exosomes recovery rate of three methods.Either in CCM or serum,two commercial kits(ExoQuick and TEI)showed higher exosomes recovery than Ultra;The protein concentration of exosomes extracted by ExoQuick and TEI was higher than Ulra,but Ultra had the most abundant expression of protein makers.Exosome samples isolated by Ultra showed the highest ratio value of particles to protein concentration.CONCLUSIONUltra is time-consuming and has low recovery rate of exosomes,but this method has less influence on exosomes protein expressin,so it's more suitable for those researches like proteomics research which need higher protein purity of exosomes.However,Ultra is also not always applicable to clinical samples due to the large volume of required starting material.So in some exoRNA research,when analysis is less influenced by contamination of proteins,ExoQuick and TEI may also good choices for their high extraction efficiency.Part 2 Different Isolation Methods of Exosomal RNA Influence the Yeild and Distribution of Exosomal RNA from Cell Culture Medium and SerumOBJECTIVETo extract CCM or serum exosomal RNA combination of the methods of extracting RNA(Trizol-LS Reagent,SeraMir,HLR,TER,miRNeasy,exoRNeasy Kits)and methods of extracting exosomes(Ultra,ExoQuick,TEI kit),comparing the concentration and distrbution of exo-RNA from different methods,and providing suggestions for the choice of exo-RNA extraction methods.METHODS1.Ultra and Trizol-LS or SeraMir kits,ExoQuick-TC and SeraMir or HLR kits,TEI-A and TER kits was combined to extract exo-RNA from CCM;Ultra and Trizol-LS,ExoQuick and SeraMir or HLR or miRNeasy kits,TEI-B and TER kits,exoRNeasy kits was combined to extract exo-RNA from serum.2.NanoDrop 2000 spectrophotometer was used to assess RNA concentration and evaluate extraction efficiency of different methods.3.Agilent 2100 Bioanalyzer with RNA 6000 Pico Kit was used to determine the RNA yield and size-distribution of exo-RNA extracted by different methods.RESULTS1.Among the methods extracted exo-RNA from CCM:In terms of RNA concentration,combination of ExoQuick-TC with SeraMir kits showed the highest extraction efficiency,which was followed by TEI-A with TER kits and ExoQuick-TC with HLR kits.However,for RNA distribution,high quality small RNA bands without longer RNA contamination can be found in ExoQuick-TC with HLR kits and TEI-A with TER kits.Ultra with Trizol-LS reagent or SeraMir kits,ExoQuick-TC with SeraMir kits presented not only the main peak around 100nt but also some longer RNA species including 18S and 28S rRNA.2.Among the methods extracted exo-RNA from serum:ExoQuick with HLR kits,TEI with TER kits and exoRNeasy kits showed the higher RNA concentration.In terms of RNA distribution,exoRNeasy resulted in the highest yield and most narrow size distribution pattern of small RNA.ExoRNA in ExoQuick with SeraMir or HLR or miRNeasy kits showed measurable bands around 100nt,but the stripes of which were not as strong as exoRNeasy kits.Ultra with Trizol-LS reagent and TEI-A with TER kits showed no obvious bands of small RNA in exoRNA samples.CONCLUSIONIn CCM exo-RNA,ExoQuick-TC with SeraMir kits showed the highest exoRNA recovery and presented dispersive electrophoresis strips not only in small RNA but also in long RNA area,it may be more suitable for total RNA research.TEI-A with TER kits and ExoQuick-TC with HLR kits showed high exoRNA recovery and also small RNA bands with higher quality,indicating superior methods for CCM small exoRNA research.In serum exo-RNA,ExoRNeasy showed high recovery yield and narrow well-size distribution pattern of small RNA,it is an improvement and fast method which could be easily adapted to clinical laboratory.ExoQuick with HLR kits can be treated as alternative methods to extract exoRNA due to the highest RNA recovery among these routes and visible bands in small RNA area.Part 3 Different Isolation Methods of Exosomal RNA Influence the miRNA Profile of Exosomal RNA from Cell Culture Medium and SerumOBJECTIVETo identify miRNA expresion profile of exo-RNA extracted from CCM or serum by different methods by means of High throughput sequencing and Realtime fluorescent quantitative PCR.METHODS1.Three exoRNA from CCM isolated by Ultra with Trizol-LS,ExoQuick-TC with SeraMir kits and TEI with TER kits were used for RNA library preparation and high throughput sequencing.The RNA constituent ratio and miRNA profile were compared between exoRNA extracted by different methods.2.Realtime fluorescent quantitative PCR was used to detect the expression level of miR-16,miR-21,miR-27b and miR-101 of exo-RNA extracted by Ultra with Trizol-LS,ExoQuick with SeraMir or HLR kits,TEI-B with TER kits and exoRNeasy kits.RESULTS1.High throughput sequencing analysis of CCM exoRNA profiling revealed that RNA content was similar,but some difference truly existed:Ultra with Trizol-LS mainly contain rRNA,when tRNA was the main RNA in ExoQuick-TC with SeraMir kits and TEI with TER kits.Besides,miRNA profile showed higher correlation coefficient between two kits methods.We also found more clean reads and higher proportion of miRNA in two kits methods than Ultra with Trizol-LS.2.Realtime fluorescent quantitative PCR revealed the variation of miRNA expression among different methods.ExoRNeasy kits perform best in MiR-21 and MiR-27b but worse in MiR-101;All miRNA from TEI-B with TER kits showed moderate expression among methods;Ultra with Trizol-LS,ExoQuick with SeraMir or HLR kits showed changeable profiling irregularly.CONCLUSIONAmong three mehods used for exo-RNA extraction from CCM,ExoQuick-TC with SeraMir and TEI with TER kits is preferable to prepare exoRNA for small RNA sequencing,and may have more advantages for miRNA sequencing.The similar results between two commercial kits could be explained by the more similar experiment principles of them.The differences of serum miRNA expression between different methods may be caused by different isolation methods have different affinity and performance for specific miRNA.