Hello everyone. Welcome to our Permega webinar, New advances in extracellular vesicle micro RNA research using high throughput affinity capture. My name is Eric Vincent and I'm a Senior Product Manager here at Permega and I'm happy to welcome you to our webinar today. Before we get started, I've got a few things to go through with you to help your engagement and enjoyment of the webinar as you're interacting with the web interface. It is fully customizable and you can move and change the windows around so it best suits you. A couple things I'd like to point out to you. We will be having live Q&A after the webinar answering questions that you pose while our speaker is talking using the Ask a Question box. There are also resources for you to look For more information, both other resources and information we have about exosomes as well as a previous webinar on demand from one of our senior scientists, Michelle Manderkar on micro RNA extraction. So there's lots of resources here. And also at the end of the webinar, we do have a survey where we ask for some feedback and we really do appreciate it when you fill those out because it helps us to continue to evolve our webinars so that we offer the content and things that you wish to hear and engage with. So as we move forward, I do have one ask for you. We do have a polling question and that's really the kind of understand where you're coming from today joining the webinar. So if you could take a moment and please reply to the survey here. What is your interest in exosomes and micro RNA? Are you and learning about it for a future project a year or so from now? Are you currently working with exosomes but looking for better methods for exosome purification? Are you looking for methods that are more sensitive in lysolating exosomes for precious samples or are you looking for higher throughput methods for exosome processing? Or are you just learning more about exosomes for your own personal development right now? So if you could take a few minutes and just give us a little feedback kind of a where you are standing today, I'll very much appreciate that. So we've got some people answering. I'll let it wait for a minute or two here while some other folks fill out. So we're getting some submissions, give you just another 30 seconds or so and then we'll continue forward with the program. All right. Well, thank you. For those of you that have submitted, we're going to go ahead and see kind of where everyone stands. So we've got a a good mix, people learning about future projects, personal development, but then some people actively working with Exosomes. And so hopefully you're going to find the content that we provide today engaging and interactive. So what I would like to do now is introduce our speaker for today, Professor Greg Rice is our joining us today to talk about a collaboration between Innovic and Promega. Professor Rice is the Chief Scientific Officer of innovic Limited precision Diagnostic Company based in Melbourne, Australia. Currently, he holds a professional appointment with the University of QLD, where he has also served as Director of the Center for Clinical Diagnostics and Deputy Associate Dean of Research in the Faculty of Medicine. Greg has over 35 years of experience as a medical researcher within academic institutions as well as 15 years in private and public biotechnology companies and senior executive roles focusing oncology, perinatology and clinical translational research. Over the past 10 years, his research and development interests have focused on the role of extracellular vesicles in health and disease and their application as biomarkers and therapeutic interventions. So with that, I will turn it over and welcomed Professor Greg Rice. Thanks Eric for that introduction and welcome everybody to today's webinar, the aim of the webinar. Is to make your. Research with extracellular vesicles easier, more targeted, and translatable. The field has changed dramatically over the past decade, and it's opportune to reflect on the EV isolation methods that we are using in our research and how those methods may impact on data interpretation. One of our first. Publications on extracellular vesicles was over 10 years ago and we were isolating extracellular vesicles from cell conditioned. Media. Using differential centrifugation, filtration and density gradient centrifugation, it took more than 25 hours to prepare EVs from 12 samples and to deliver this type of data. Western blot analysis of the canonical EV associated proteins and in this case a targeted protein placental alkaline phosphatase and ultimately we pulled those density gradient fractions to identify a biomarker present in those intracellular vesicles. We can isolate EVs from 96 and 90 minutes to deliver similar, if not better. Data so. This is one major change that has occurred over the past decade, our capacity to process samples and isolate EVs in a timely manner. While the field has advanced considerably, many researchers are still. Isolating using. Isolated methods that are slow, tedious and low throughput. And while there might be specific reasons for doing so, these methods are not viable for many preclinical and clinical applications such as biomarker validation studies and diagnostic applications. They cannot be directly transferred into clinical pathology workflows to process the perhaps 4 or 500 samples that need to be processed in a day as a in vitro diagnostic. And this is an issue that I want to focus on today, the development of a fit for purpose EV biomarker discovery and diagnostic development platform as Everett mentioned. I'm Greg Rice, a Chief Scientific Officer of Innovc Australia. We are an exosome diagnostic and therapeutic company and working together with Promega, we have developed EV enrichment and RNA protein isolation platforms that deliver high throughput sample processing. We can now process up to 4 to 500 samples per day. That's more than 150,000 samples. Per year and this is. What I want to focus on today and there are really three areas that I want to highlight. Firstly, to talk a little about some of the challenges and solutions in working with extracellular particles. Secondly, our approach to resolving some of those challenges. And then finally to work through some reference studies where we have applied high throughput EV isolation to identify extracellular vesicle associated micro RNA and the two case studies that we'll look at the relate to breast cancer and ovarian cancer. So let's begin with some of the fundamentals of extracellular vesicle biology. The International Society for Extracellular Vesicles defines extracellular vesicles as a generic term for particles naturally released from cells that are delimited by a by lipid layer and cannot replicate. EVs are released from all cells and heterogeneous population of vesicles. Raising. Ranging from size of 30 nanometers up to several microns, they are of diverse biogenic origin, can be formed from the endocytosis of the plasma membrane and inward budding and cargo packaging to form multi vesicular vesicles that subsequently fused with the plasma membrane and release exosymes. They can also be performed via the exocytosis of the plasma membrane. E VS are able to deliver effector molecules to both proximal and distal cells by membrane fusion by micro PIA cytosis endocytosis or receptor mediated activation of tiger pathways. Importantly, they encode information about the cell of origin. Two years ago we prepared a scientific statement for the Endocrine Society to help guide EV based research in the domain of endocrinology and we proposed that EV signaling cell to cell communication may be considered as part of the endocrine system, being that they are circulating messages that regulate cell and body function and respond to challenges to homeostasis. One of the important points that we wanted to make in that statement was that while EVs can be captured from Biofluids and their messages read and related to cell status, the heterogeneity of EV populations must be considered in interpreting these changes and the the messages read will be affected by the methods used to isolate those extracellular vesicles. And we're really left with the question of which vesicle are we targeting and what is the message that we're reading. The situation with the complexity of EV heterogeneity continues to evolve, and last year Yepsen and colleagues published a very elegant review on the heterogeneity of extracellular vesicles and extracellular particles, in which they referenced 18. Different. Extracellular entities that may be present in biofluids and they noted that the extent of Co isolation with other particles varies with the isolation method used. So for heuristic purposes, if we consider A2 dimensional display of particle density versus particle diameter, this point becomes much clearer. For. Precipitation and 100,000 G sedimentation isolation methods. They are somewhat agnostic with respect to the particles that they isolate. Size Exclusion methods partition particles on hydrodynamic diameter and isolate particles of similar size but variable density. Density gradient isolates particles based on their similar densities but not their size. One of the promising approaches for enriching extracellular vesicle subpopulations is to target surface epitopes on the vesicle surface. The effectiveness of this approach clearly will be dependent upon the level of expression and specificity of the target epitope. But it becomes clear that the method used for isolating EVs impacts on downstream analysis and their associated cargo and biomarkers, and we need to be very careful in considering the intended use of the EV isolate preparation to provide a high level. Overview. Of the extracellular particle landscape grain familiar and regularly report findings related to membrane bound extracellular that are present in biofluids. What is under reported is the presence of non membrane bound particles, a diverse group of particles that may be present in biofluids and Co isolate with EV preparations. If the intended use is to address questions about EVs, or indeed subpopulations of EVs, then consideration should be afforded the potential Co isolation of a more inclusive cohort of extracellular particles. And really this is where more targeted approaches for enriching subpopulations of EVs that do not rely on the common or shared features of isolating EVs may be of benefit. So key points around this section of the webinar isolating particulate fractions from biological fluids using methods based on hydrodynamic size, density or charge result in heterogeneous isolates. The preparations contain a mescellony of both membrane bound and non membranous particles. This may hinder or confound robust characterization of the EVs that we are targeting, that we're interested in and also the meaningful assessment of their specific biological activities. Indeed, we're not sure. Which particle? We're focusing on or isolating or to what proportion that may change with the different isolations methods used, isolation of tissue specific or enriched. EVs. They cannot be achieved using methods that rely on features that are common to all P or all extracellular particles. If we're separating on size, we're not going to be able to effectively separate extracellular vesicles from extracellular particles. Isolation methods that are not based on common features are required to provide a better understanding of the role of EVs, and a promising approach here is to target basically surface epitopes. Ultimately it depends on the intended use of the EV or EP preparation and the selection of the isolation method used. I now want to move on to our approach to addressing some of these challenges. And the approach that Promega and Innovic have taken to provide more effective tools in isolating EVs is to focus on affinity matrix capture methods that target vesicle surface epitopes And an integral. And an integral component of this development process has been to clearly define user needs and the performance characteristics of the product. And these include being a rapid, simple and cost effective method. The isolates need to be compatible with downstream analysis for RNA and protein characterization. They need to be able to enrich the target population of EVs and importantly, to be reproducible. They need to be scalable to be able to be run manually in medium and high throughput and fully automatable, allowing them to be compatible with workflows in routine pathology laboratories and perhaps ultimately need to be compatible with CGMP production as required. These user requirement specifications were expressed in quantifiable terms to allow meaningful evaluation of the performance of the product. In particular, we were seeking to develop products that had an average sample processing time of less than two minutes. Could process up to 400 samples a day. Cost competitive with other methods have appropriate coefficients of variation in turn in Tri. Run use low. Sample volumes Around 500 microbes of plasma and perhaps 2 mil of cell condition medium generate suitable amounts of total RNA and protein to allow downstream analysis be scalable and compatible with commercially available kits. The approach that we took was a simple approach with the intended use to enrich for a subpopulation of EVs in biofluids that contain a heterogeneous. Population of particles. Both membrane bound extracellular vesicles and non membrane bound particles that may have overlapping size, density and charge characteristics. We built a three-dimensional affinity capture matrix on a paramagnetic bead. This allows for manual semi automated and fully automated isolation of EV associated RNA and protein cargo and particularly this was aimed at biomarker discovery and ultimately diagnostic development. The product that we developed is called Exonet and this is the manual workflow depicted here. Bio. Fluids of between 100 and 500 microliters are mixed with 15 to 30 microliters of exonet and incubated for 15 minutes. The Exonet Exonet beads are immobilized by magnets and washed and the captured EVs are then ready for either on beat analysis using for example FTIR fax oraliser or on bead lysis for the analysis of EV associated RNA and protein representative data for the manual isolation as shown here on the left for western blot running canonical EV associated proteins and on the right for EV associated micro RNA. The data for CT values for exonet are shown in yellow. The other bars represent other commercial products currently available. Similarly for a group of RNA data presented for exonet in the yellow and other bars in for the commercially available kits. For me that has also implemented the exonet isolation on the Maxwell RSC platform and following Isolation of EVs using exonet the beads are loaded. Onto. An micro RNA plasma serum cartridge and run exemplar. CT Values for three micro RNA are shown and an application note is available from Promega. Promega has also implemented a fully automated 96 world plate format for isolating EVs using Exonet and a commercially available particle moving platform EVs again isolated using Exonet. Both the sample and reagents are loaded onto 96 row plates placed in the particle moving platform, in this case Kingfisher Apex platform and EVs are isolated in 90 minutes. The isolates are suitable again for on bead analysis or on bead license for mass spectrometry or for processing for RNA or micro RNA using RNA, seek qPCR or digital PCR. Comparing the actual performance with the design inputs, we satisfy many of the requirements in the design process. Using the platforms that we have developed, we see an average sample processing time of less than two 2 minutes per sample. We can process up to 450 samples per day. The price of the product is competitive with other B based EV isolation kits. We achieve appropriate coefficients of variation between runs and within runs. We've been able to use as little as 100 microbes of plasma for RNA and protein analysis routinely. We're using 200 microagers of plasma and 30 microagers of exonet and we've been successful using two mils of cell conditioned media rather than having to concentrate large volumes that may be required by other procedures. And we're delivering suitable quantities of total protein and total RNA. The system is fully automated and compatible with commercially available kits and the platform has been obviously. Developed with. Using Promega kits, so summarizing the key points, we've been able to demonstrate that our design outputs making our design inputs the the platform's scalable. It can be run in a manual form, high medium throughput platform and high throughput. It's rapidly, it's reproducible, can be run manually. We process up to 42 samples a day using manual operations on the Maxwell. It's in a format for 16 and 4848 sample runs. And for the. Kingfisher, we're running it in a 96 world plate format and we've been running for up to five plates per day and we now have analytical validation and clinical validation of this method. So the final part of this presentation, I want to share with you some of our experiences in using the high throughput platform for clinical samples. These two reference studies are case control samples looking at serum from breast cancer patients and also plasma samples from ovarian cancer patients. And really the objective of these studies was to isolate and analyze EV associated micro RNA. In the breast cancer study, we've used 95 samples of breast cancer serum. The cases were evenly distributed over stage 1 to 4. The isolation of extracellular vesicles was conducted by Promega and in Madison using half a mil of serum. The serum was spun for 5 minutes at 10,000 G and then processed on the Kingfisher Apex. Platform. Using the Maxwell HT Micro RNA Plasma and Serum kit, samples were then shipped to California to a contract research organization, Research DX, where they used a small RNA library prep kit and analyse the samples on a Next SEEK 500 alumina platform. Using this process we were able to identify 53 significantly differentially expressed micro RNA both down regulated in cancers and up regulated in cancers. We were able to characterize the top 20 differentially expressed micro RNA in terms of their their positive fold change and negative fold change and 26 of these micro RNA were subjected to micro RNA net modeling and identified both EV related proteins and were associated also with breast cancer related proteins. Using pathway enrichment analysis and the KEG database, we were able to identify multiple cancer signaling pathways. So the outcomes of this study we were able to successfully use the high throughput platform to identify micro RNA that are significantly expressed in breast cancer patients and with modeling able to associate those with known EV proteins and cancer related proteins. The second study considered easy isolation from plasma samples obtained from women with ovarian cancer and normal healthy women and we use 50 samples here and cases were split between stages one to three. This study was performed in Innovic's laboratories in Melbourne. The isolation We used exactly the same protocol using half a mil of plasma, centrifuging it for 10,000 G and running it on the Kingfisher Apex platform using for Amigas HT micro RNA, plasma and serum kit. The isolates were then shipped to the Australian Genomics Research facility where they used Netflix small RNA sequencing kit on the Nova CX Plus platform from alumina. And again we were able to identify significantly differentially expressed micro RNA 27 that were altered between disease and normals. The RNA we identified had been associated with tumor promoter and growth and metastasis of breast, ovarian and brain tissues. Any of these micro RNA were subjected to micro RNA net modeling, looking at over 1000 genes and associations with EV related proteins and cancer proteins again were demonstrated using GO analysis. It was reassuring to be able to identify in terms of. Cellular. Components associated with extracellular vesicles and other compartments. These samples we also subjected to RTQPCR. We processed them within Innovix Laboratory in Melbourne. Use Tekman and Quant Studio RTPCR to profile a panel of 16 micro RNA. Using this method we were able to identify a series of micro RNA that remained constant across all samples, in this case showing 3. But we also identified a single RNA out of the panel that was increased across all stages of ovarian cancer. So this simply shows the utility of the platform being able to go to micro RNA, seek data or to process them on RTQPCR platforms. So the outcomes of this study how we're again able to identify differentially expressed micro RNA in the cancer samples and relate these to known pathways involved in the disease process. So a few concluding comments of the webinar. Firstly, selected subpopulations of AVS can be enriched using affinity matrix capture and here we've used the Xnet product. This can also be customized for brain derived DVS, presented derived DVS and exercise derived DVS and we have used these in studies to date. The platform is scalable, reproducible and rapid for the isolation of EV associated analytes, protein, micro RNA, messenger RNA. It can be used in a manual format where we can process up to around 42 samples a day on Promega's Maxwell platform, either in a 16 or 48 sample format and on the. Kingfisher in a 90. Six world plate format, the latter being compatible with the requirements of a pathology workflow. The isolates are suitable for RNA seq RTQPCR and protein data independent acquisition using mass spectrometry and we've had run a number of studies now using mass spectrometry. The latest study we ran was a 440 sample study that we were able to turn around in two days. So have you have found this webinar useful and I encourage you to consider using the system. To make your. Research with extracellular vegetables. Easier, more targeted and translatable. The products that we've shown in this webinar are available from Promega and application notes are also available on the resource links associated with this webinar. So thank you for your attention and welcome any questions and feedback. All right. Thank you, Greg. We're we're back live and if you have questions, please feel free to use the Q&A box in the in the webinar interface. I do have a couple things I'd like to work with you folks before we get to the questions. I do have two questions to to pose to you folks before we get to the questions. So the first is what methods do you currently use for exosome purification and click all that that apply. Do you use ultra centrifugation, peg based centrifugation, density gradient centrifugation, size, exclusion chromatography, immunoaffinity purification, ion affinity chromatography or something else? So if you take a few moments to let us know kind of what you're currently using, if you're actually purifying exosomes currently, we're kind of curious to see what folks are are using. So we'll wait a minute or two. We got some folks voting right now and I appreciate your feedback and input. Wait just another couple seconds and I have. All right. So we've got submissions here and I'm going to go ahead and show you guys the results and we'll just see kind of where everyone stands. Not surprising that kind of the the current gold standard of ultrasound irrigation has the highest number of votes followed by size, exclusion chromatography and then PEG based and immuno Infinity purification. Thank you for that. One final question, just thinking about for your laboratory either currently or looking ahead to the next year or so, how many exosome isolations in an ideal world would you like to be able to accomplish per year? Is it a small number, less than 100, up to 500, a thousand more all the way up to and you know greater than 10,000. Just to kind of curious to hear about what throughput are you looking for right now? Are you looking for automated methods? Are you looking for low throughput automation like the Maxwell system or is manual scale and throughput appropriate for what you're doing? All right, well, I'm going to move ahead. We'll see kind of where where folks are with their throughput, some at low throughput and then kind of a sprinkling throughout all the way up to to higher levels of throughput. Thank you so much for answering my questions. Now we're going to go ahead to your question. So Greg, the first one the first question I have is from is from Wei and Wei is asking about kind of thinking about Exonet and that Exonet uses antibodies and Wei is asking how would how how does the antibodies with Exonet interact our work when you're doing downstream downstream proteomic studies and and how kind of how is that approached it approached and is that is that ever a problem in the downstream assays? Yeah. Look, thank you very much for that question. Hopefully you can hear me and see me OK. Yeah, look excellent contains a number of antibodies that are covalently bound to the magnetic bead. We, our collaborators and also some of our customers have successfully completed downstream proteomic analysis using Exonet. The process is clearly to rise extracellular vesicles that have been captured by Exonet and then use that lysate for whatever flavor of proteomics you choose. As I mentioned in the webinar, we have been using data independent acquisition mass spectrometry. We've also used. Targeted mass spectrometry using MRM and single reaction monitoring and we've we've not found a problem in terms of the dissociation of antibodies with your exonet isolate. So I think we we have sufficient data there to reassure you that the Protea mix that you see with using Exonet are of suitable quality for your work. Well, thank you, Greg. We we have another question from Raima and they're asking how do you confirm that the protein in the EV sample is exactly associated with extracellular vesicles and how do you avoid soluble protein contamination? Can affinity isolation ensure this? So the question, Eric, was I'm just looking for it now. So kind of asking about valuable protein contamination and. Yeah, yeah. Look, look the way we've developed vaccine it is to to isolate EV associated proteins and the the targets that we have used obviously membrane bound membrane associated proteins to capture the EVs. It's it's possible that if you do have soluble protein that are recognized by those antibodies, they could contribute to your downstream analysis. You've also got to remember with respect to soluble proteins, when you're processing samples using Exonet, there's an extensive washing period to remove any soluble proteins from your islet, something that also is easily checked. Yeah. And and I I would, I would add also in addition to that Innov CAS shown some data where there's they specifically look for soluble protein markers via mass spec versus more extracellular markers And and we definitely have seen that those kind of serum derived markers are much decreased in the samples purified with exonet compared to other methods. So that kind of also addresses that. Another question again looking at E VS from blood. So the majority of E VS in the blood from plasmid serum are derived from red blood cells and platelets. EVs isolated by exonet do not discriminate EVs released by blood cells from EVs from carcinoma cells. So regarding to breast cancer, ovarian cancer studies, how do you know the micro RN as amplified by PCR from exonet EVs are actually derived from cancer EVs? Yeah, look great. Great question. Exonet was designed as a pan capture tool. The subpopulation of EVs. That it. Captures is really. Defined by 10 different protein epitopes that are present on the surface of EVs and those epitopes were selected on the basis of either known EV or tissue protein profiles. So we are capturing with XN at a broad cross-sectional population of EVs The in terms of being able to perhaps refine that Exonet is also customizable and it can be customized to customers requirements if there are highly. Expressed epitopes. On the cells or tissues of interest the the composition can be changed. For example, we have developed another version of Exonet called Neuronet that targets proteins that are expressed to cell surface proteins that are expressed by brain cells. Using neural net, we isolate a different subpopulation of extracellular vesicles that display a completely different proteomic and RNA profile. So it can be tuned to customer requirements and it really depends on on what your intended use is for the preparation that you're you're seeking to analyze. Yeah. So, so it really kind of depends on the question. It exonet the, the current product is really designed to isolate all exosomes and then depending on interest more targeted approaches could be taken using the same technology. OK, on to the next question, can exonet be used? Most of what we talked about today was based on plasma derived exonet. Can it be used to identify EVs from other biofluids besides plasma? Yeah. We customers and collaborators have used it on a number of biofluids, including obviously similar plasma. We've used it on saliva. We've actually just recently one of our calibrators has published a paper looking at small extracellular vesicles in saliva. A copy of that manuscript is available on the resource section for this webinar. We've also used it for urine and cell conditioned media. So it can be used by for a number of different biofluids. It can be used as we've mentioned for either on bed analysis using techniques such as FTIR, even immunoassay and also OXY flow cytometry. The on bed lysis can be used for ELISA, western Blot, obviously RTPQQQQPCR mass spectrometry and RAC. So versatile and certainly suitable for many biofluids. I think the good thing about the before I let you go on Eric, the good thing about the SO condition medium and using Exonet, we've been able to successfully use Exynit on as smaller volumes as one mil of cell condition medium for RNA profiling and protein profiling. This avoids what's currently the practice of collecting fairly large volumes of cell condition medium, concentrating them down and then then using other methods to try and isolate the EVs. So it reduces the workflow involved and that's essentially a one step process on small volumes. OK. Next question, Greg, this is actually one that I'm quite interested in. How do you want, how do you quantify the number of E VS captured? With Exxon. OK. That's, yeah, that's a really good question, Excellent question. Exonet was designed for. Capture. And downstream analysis of EV associated biomarkers, it was not designed for capture and release. So the way that we've been able to estimate EV capture using exonet is by depletion analysis and nanoparticle tracking. So by determining what's in the input solution and what remains after Exonet treatment, we can get an idea of the number of EVs associated with the Xnet capture process. The other way to do it is obviously to look at some of the canonical biomarkers of EVs and try to relate that then back to a semi quantitative estimate I guess of how many EVs might be there. OK, excellent. Next question, Oh. There's a nice question there about bacterial contamination. You got that one, Eric? Sometimes my EV samples get bacterial contamination. Oh, yes. Sorry. Yeah. Actually, I. Missed that one, yeah. Yeah, yeah. Really, really timely question. The the collaborator who has just published this paper on saliva, they actually have the same problem as as you do of bacterial contamination and in in relation to for example, looking at periodontitis in the paper they actually look at bacterial contamination using exonet compared to other processes and what they found is that exonet dramatically reduces the contamination by bacterial components. So I direct you to that paper which is available on the resource side of this webinar. OK, another question here from looks like Shirani, is there any comparative study of ultra centrugation and exonet isolation in terms of the number of EVs isolated? Probably, probably not in terms of the number, again because we're using depletion analysis to to estimate the numbers we're capturing EVs. We're not the first not designed for capture and release, but a number of our collaborators and customers have compared differential centrugation, so ultra centrugation and density gradient centrifugation in in with respect to the contamination for example by albumen. And what they're finding is a 5 to 6 fold reduction in the contamination Co isolation of albumen using exonet compared to that particular process. And the way they estimated that was by mass spectrometry looking at peptide intensity from the same collaborators. They've also looked at the Co isolation of Apollipoproteins and comparing those techniques of as you know ultra centrugation versus affinity capture using mag beads. What they report is a six to. Tenfold less. Contamination by APOB and which was relevant to this. Particular study. So they're the types of comparisons that we have between the various techniques. If your work specifically focuses on trying to understand the the functional biology of exercise and you need them to to be intact and release, we do have another product called XO ACE which we've developed in our therapeutics division to isolate bulk exosomes. So that's also available. OK. We're starting near the end of time, but I've got a couple more questions here. The next one was, this is an interesting one. Do you? Do you or does anyone provide high throughput EV isolation service or services? Yeah, look we've we've now validated the the high throughput platform extensively and what we primarily offer that to biotech and pharma. We do have a high throughput in the isolation service here available in Melbourne, Australia and also in Irvine, CA through a CRO Research DX. We're currently also seeking a partner in Europe to provide such a service. These facilities are able to turn around 480 odd samples a day and the minimum batch requirement for that service. Is around about 96 samples per job. So yeah, it's available. Excellent. One thing I will add to that as well as that Permega actually with the purchase of our products, we have scientists that will help laboratories automate extractions including the Exonet we currently also available in the in the resources we have a couple application notes. We've automated this on the Kingfisher Flex and Apex and we also have experts in all of the major liquid handlers available. So if someone is looking to process their own samples but really needs help programming that, there are ways that Per Mega can can help assist with that as well. Let's see, Let's see if we have one more question here. Actually the last one let let's switch to something we didn't really talk about all that much aside from a couple questions. Thinking about protein isolation methods, because I I do know that a lot of folks are looking both at RNA and protein. What are the available EV protein isolation methods? OK currently we we have an in house exonate isolation process that basically soluble lies as the the membrane of the EV allowing us then to recover the protein that are. Associated. With EVAS I I know we're keen to to work with our partners on developing bead based approaches to recover and fully automate the the protein process. Protein isolation analysis. Process but at present the application note that we provide. With exonet. Documents in house process that works very well with the mass spec platforms that we've utilized for Western Blot. And also for Elijah analysis. All right. Well, excellent. I'm sure probably that's very keen to develop those techniques as well. Definitely. We definitely have some protein expertise at Promega and stay tuned for there were more developments on that front from Formaga as well. It looks like we're nearing the end of time and I believe we've had, we've answered all the questions. If if anyone attending has anything else, please feel free to go ahead and either send us an e-mail or ask in like the Q&A chat we go back I I kind of have one. One final thing for everyone and that's to offer our thanks. And as we end the webinar, we do have a really short survey on the webinar, your experience. Just a couple quick questions to help us as we think about our next webinars and scheduling continuing content that you'll find useful for your research. So with that, I will say thank you all so much, those of you in the middle of the day or in the evening spending time with us. And we look forward to seeing you at the next Primego webinar. Thank you so much. Have a good day. _1714204028612