Hi, everyone, and welcome to today's webinar on precision and veterinary nucleic acid purification optimizing complex samples. I'll be your moderator for this event. My name is Michael Isaac, a segment marketing manager here for MEGA. We're excited to have our guest speaker share and present on topics working with diverse sample types in animal health and the role of accurate and efficient purification and downstream applications. Just a couple of housekeeping items before we begin. I would like to cover that on your screen there are multiple windows, all of which are movable and resizable. So feel free to move them around and get the most out of your desktops desktop space. In this webinar, we have many ways to be interactive. Submit a question at any time during the during the webinar. We'll answer these during the live Q&A. In the resource library. There's a list of helpful materials, which also includes a copy of today's presentation. Feel free to download any resources or bookmark any links that you may find useful. After the presentations, there will be a quick survey. Please take a moment to answer these few questions. We'd really appreciate any feedback you may have. You're also welcome to share the webinar with any colleagues so they can learn more. Next, I'd like to introduce our speaker for today. But before we get into that, let's get it into just a couple quick polling questions where we're interested to hear and learn a little bit about the work that's being done here in the community for vet animal health. So what is the most common sample type you typically work with for sample purification in animals? Give it a few minutes for everyone in the audience. See 11% answered. Some of the options we have our blood, saliva, hair and fur, tissue biopsy and microbiome. OK, still seeing that number going up, got 30%. OK, OK, I see you in the chat as well. We deal with feed. Very cool. OK, so we have it at 36% answered. We'll just take a quick look. So it looks like a majority, those in the in the audience are working with blood and and microbiome types, which is interesting. Maybe we'll keep this in mind as we move forward with this presentation. On to the next one. What are the biggest challenges you face with nucleic acid purification? Is it anything to do with contamination, low yield cost, potentially the time or even another? OK, see roughly similar 30% answer. So we'll move and look at the responses here. It looks like a majority of people are seeing low yield. So we'll we'll keep that in mind and look to tailor our our feedback as a result of that. Now let's get into our guest speaker for today. We're here and excited to welcome Patricia Irishi. She's a Senior Scientific Account Advisor at Promega Corporation with a background in gastrointestinal research on companion animals. Patricia has significant experience in intestinal permeability, microbiome studies and use of micro RNA as a biomarker. Patricia holds a PhD from Texas A&M University and a vet medicine degree from Mato Grosso del Sol Federal University and. Hello everybody. My name is Patricia Edishi. And before we start, I would just like to thank Pramega for the opportunity to talk about optimizing complex of urinary samples. So Michael already gave a little bit about me, but just to rainforest, I'd like to say that I have 3 personalities. I have Pat the veterinarian, Pat the scientist, and then Promega Path. I graduated from that school in 2016 in Brazil at the Federal University of Mato Grosso Dusou. While in that school, I had the opportunity to study for one year at Iowa State University and intern at the USDA as a Veterinary biological Control intern. I then want to pursue my PhD from Texas A&M University Gastrointestinal Laboratory where I researched the microbiome and biomarkers in dogs with chronic anthropathy. I have joined Permega team in 2022 and it's been a pleasure to help scientists all over the world since then. So during my entire professional journey, I have worked with multiple and diverse veterinary samples. Thinking back, it's kind of crazy because I work with nematodes from alligators, Texan cattle, and blood, feces, tissue, and FFB samples in both dogs and cats. So hopefully today we can share a little bit about how I troubleshoot one of my most challenging sample types. As you probably know, veterinary samples include a variety of samples from multiple hosts and diverse applications. Here we illustrate a small world of possibilities from working with companion animals, production animals, wildlife and being able to collect from clinical samples all the way to food products such as meat, dairy and so on. The applications of why you would extract nucleic acid from the samples are also endless. It could be from path, it could be for pathogen detection research or new research of new biomarkers, sequencing, genotyping and many more. But let's talk about the real challenges of working with varinary samples that we see every day. So in first place, I added availability. In my experience, varinary samples are way more scars than human samples. We don't have huge biobanks like human hospitals do. And have you tried collecting blood from a Chihuahua? It's really a nightmare. So because those samples are so precious, they're kept store from previous studies. And more often than you can imagine, we find ourselves with leftover samples that you have to work with whatever you have because that's all you have, right? And then the third challenge is probably cross contamination as it's very hard to collect feces that haven't touched the floor or do a stereo collection in a farm. Lastly and possibly the biggest nightmare of all scientists is degradation. Nucleic acid degradation can happen for seven reasons and are very common in veterinary medicine because not always a preservative or optimal conditions are available. Here I highlight the importance of quantity, quality, integrity, and accurate quantification for several downstream applications such as amplification and sequencing. As you can see, quantity and quality is not as important for amplification methods such as PCR and qPCR as it is for sequencing, so this is something you will have to keep in mind when choosing what is important to you and your research. I will talk about troubleshooting samples using my own experiences as an example. So my first project in grad school was to evaluate micronate 29A as a potential biomarker for intestinal permeabilities in dogs with chronic enteropathy. So through RTQPCRI was able to get this data. So as you can see, this was pretty straightforward. They were old samples, but I used amplification. So honestly, quantity and quality and integrity wasn't much of A concern to me at the time. So for the next step, we couldn't help but wonder if there were other micronase that could be used as biomarkers for gastrointestinal diseases in dogs. And to do so, we decided to do an untargeted RNA seek to find new micronate biomarkers in dogs with chronic anthropathy. Our goal was to collect fresh fecal samples from both healthy dogs and dogs with chronic anthropathy, extract the RNA, check the RNA integrity and quality, send samples for untargeted RNA seek, and then help hopefully find a novel micronate to be used as a biomarker. But then very soon I hit my first roadblock. One of my first roadblock was, you know, getting a good urna from all of my samples. They were fresh, so I was positive. All of them should give me good you, right? Wrong. I spent a solid couple months trying to find the best extraction kit for my samples. And what I learned is that for fecal samples, the most important step is definitely to do beating. As you can see on the graphs, the same sample extracted with two different kids, can you complete different report for the patient. So optimizing and finding the basket was a priority as I didn't want to report a patient to be negative if they were positive and vice versa. When I finally found a good kid and went on extracting all of my samples, then I had my roadblock #2 as you probably imagine, this was my pH D roadblock after roadblock. My second challenge was really freeing myself from the information I learned in textbooks and through review articles. I always thought that before we sequence our samples, we should check the RNA quality integrity using the Bio Analyzer, and I'm sure you've heard that too. You know, we should expect really high rind values as shown here on this image. So I spent some money to take all my RNA to our core facility and use their tape station. And this were the values I got. As you can imagine, I was desperate. I was thinking what should I do now? Like I won't be able to sequence this, you know, I have a deadline, I'll have my samples, I already extracted all. So what should I do? I cannot tell you how many times I re extracted and try to troubleshoot my samples before I accepted the fact that theses are thesis. You know there will always be degradation and my rinse car was never going to be perfect. When you are troubleshooting your samples, understand the nature of your samples. For example, it will be more challenging to get a long sequencing data from FFB and fecal samples versus the clean plasma sample, and there's not much you can do about it. Troubleshoot your sample preprocessing. You may need to include a step of BE beating or protein ace K digestion. Re evaluate your quality control methods such as Nanodrop versus Qantas using spectrophotometer versus fluorescent probes for MEGA actually has a great recorded webinar on quantification of nucleic acid as those two methods can vary depending on your application. But most importantly, use high quality reagents that will ensure you high quality nucleic acid extractions and the end. I did send those samples with horrible written numbers to sequence and I'm happy to report that we were able to identify 10 differentially expressed micronase and dogs with chronic anthropathy. I will not show the data as we haven't published yet, but having a highly degraded sample didn't prevent me from getting the data I needed because I understood the circumstances of my samples and optimize using the best reagents. So after a method is chosen, how do you improve reproducibility and optimize your time in the lab? As you know, manual extractions can be lengthy and quite frustrating, especially with pieces. And I've seen that many of you are doing microbiome work, so I hope you guys can relate. Because no matter how good you are pipetting out only the supernatant, there's always some small particles that clogs the columns. There's also a high chance of cross contamination in between samples if you're not cautious manipulating the tubes columns and discarding the flow through appropriately. And I had still haven't discovered why the human influence the quality to, you know, some people are just DNA whispers. So I wanted to highlight what came to be my secret weapon and actually one of the reasons I'm at the Permega team. During my troubleshoot. I came across the Maxwell RSC systems. The Maxwell RSC system is an extraction automation that speeds up your lab's entire workflow. Maxwell efficiently processes various sample types, greater reducing time and compared to manual methods. Maxwell is not a liquid handler though, so it is a we consider as a particle mover as he uses magnetic beads to purify DNA and RNA. One of my favorite parts about the Maxo is the fact that he has a pre filled cartridges. So you'll have to spend the time pipetting reagents and minimizes reagent waste, allowing you to extract as little as one sample if you need to. So and then the cherry on the top is that it's super user friendly. So he has a touchscreen interface and very intuitive to use. The Maxo really allow me to streamline my extractions. You know, it saved my time twice, once by reducing my hands on time and then twice by allowing me now to train my student workers to do the job because it was so easy to use. You know, so it saved my own time and also saved me because then it was easy to train my students. And on top of all of that, I got super reproducible data and high quality product to move on to my downstream applications, in this case RNEC. I would also like to highlight a very cool and Permega team of Permega, which is the scientific applications team. They are a global support team that adapts Permega products to customer needs through consultation and experimentation. They are experts in many areas and I actually work with them to troubleshoot many of my experiments and one of the data you guys will see here. I wanted to highlight a couple of the products they did so you understand how this team works. This one was using pig oral fluid collected from 2 ropes for detection of viral and bacterial pathogens in pig Hertz. So you know the sample type were oral fluids from 2 ropes. The application was pathogen detection and the materials were usable. The Maxo RC pure food GMO and authentication kit for the conclusions, we concluded that we were able to extract total nucleic acid from the shoe ropes and from 200 microliters of pig oral fluid. And then for to do that, we use the Maxo RSC Pure Few GMO authentication kit or alternatively if you want to use a manual kit and we have the reliable prep blood GDA system. This is another project that they did, which was automating DNA in RNA purification from canine feces, serum and buccal swabs using the Maxwell RC instrument. So we use dogs and then for the sample types, we have canine feces, serum and buccal swabs materials would use all those three kids. So depending on the application, you can use the RC fecal microbiome, the micronate plasma and serum and then the RC buccal swab. And here is the result. The conclusions for the buccal swab, you can see that for DNA purification, DNA will successfully purify using the Max ORC buccal DNA swab. And then all samples were amplified by Q PCR targeting the IGF one locus using GO Tech Q PCR master mix. And then no amplification was observed on the negative controls, confirming this project was this project was a success. The conclusions on the serum study was that the Maxwell RSC micronate plasma and serum kit is compatible with canine serum samples. So here we were evaluating micronate 29A and serum samples from dogs. So you can see that this was one of the projects I collaborated when I was a graduate student and we were able to use this kit to extract from really low amounts of serum only 200 microliters. And the conclusion for feces, this is a new applications that we have available for you guys. But DNA from 10 are carbo canine FICO samples and the blank control were purified using the Max of FICO micro microbiomes DNA kit. 5 microliters of each DNA sample was amplified in a 20 microlitreal reaction using Georgia Lambia specific primer. So here we had eight known samples positive for GRGM that we tested using Perminga's extraction kits and also master mixes and we were able to confirm those samples were actually positive and negative accordingly. So this was the last application in canine samples. We they also do pretty cool projects that are not just domestic and production and domestic and animals. And this one was a high molecular weight DNA purification from bovine samples. So they use the domestic bovine cattle as an Organism. Sample types were blood, hair, tissue, ear punches and Tzu tubes. And we just mainly use for pathogen detection. We were able to highlight the Wizard high molecular weight DNA extraction kit. This is a manual kit that you could use for high molecular weight. So here is the conclusion on the blood extractions. You can see the DNA was successful extracted from 300 microliters of bovine blood using the Wizard High Molecular Weight DNA Extraction kit. And you can see that the DNA was run on 0.75% aggro's gel for 16 hours using a piping pulse and then Lambda, PFG ladder and Chef DNA size stander were used as markers. So we were able to use our kit to do this experiment as well. We also have some conclusions showing the hair data that high molecular DNA was successful prefy from bovine hair using that same kit for the BFG analysis, 500 nanograms of DNA per sample were run on the aggro's gel for 16 hours. So you the same data as the other one. We were able to get from here samples of cattle And then lastly. We have the tissue ear punches where we also analyzed the DNA from those bovine umbilical cord and placenta and ear punches and TSU tubes. And same as the other two, we were able to conclude the experiment with really good data showing that you can use that kit for diverse diversity of urinary samples. We do have additional resources for you to look up after this webinar with links to brochures and the application notes I showed you. So they all be available to you for you to download and take a look. And lastly, I would like to highlight all of promenade programs and promotions going on right now and I would like to encourage you to check them out. Our newest one is the wizard in your lab. So every lab has a lab with and the scientists will tackle those challenges and support breakthroughs and keeps everything on track. So you can actually nominate your lab wizard today and both of you could win a prize. So just really encourage you to go and see all per mega promotions going on right now. And with that, I guess I'll just open to questions. Hey, Pat, great question. This, this came from the audience and, and I'd like to also integrate a lot of the sample types they're working with, whether it's microbiome and blood, just to get a your perspective on their work. One thing they were interested in and I'd like your perspective on is how quickly post collection should samples be processed? And do you feel like that time between sample collection and then actually running the samples, Do you feel that time has any potential limit where it's, it's in fact introduced to RN Asus? And what if anything in terms of preservation should be done up to that point of, of processing like in your in your work, did you use any flash frozen or stored in any RNA preservation reagent during your, during your studies? Yeah, I guess it would depend of what I was extracting from. For feces, I'd say most of them were not preserved other than being frozen. And -80 I was also using archival samples. So it's really hard to have everybody, every study save their sample in the same way and a factor and result. So I guess the standard for fees is just frozen freezing them a -, 80 issue, though a lot of people do put them in RNA later if they know they're doing RNA work. But we do know that after a year being frozen, no matter what, there's going to be high degradation on those samples. But I would just really want to. I don't know if Sarah has anything to add to what I just said. So there are some different types of fecal stabilization tubes that we've worked with in scientific applications group. And so if you are looking to try to stabilize the RNA profile or for example, like a bacterial profile, if you were doing a microbiome study, you might want to look into using some of those tubes. Just just as a a follow up question as well, we saw most of the audience working with blood and microbiome based samples. Do you feel there are common contaminants or sources of impurities to watch out for, whether it's during DNA or RNA extraction that might, you know, like like you mentioned in your data would showcase with low rent scores or something along those lines or just low quality RNA? Yeah. I mean, it's what I said. It's really hard to get a really good integrity and DNA or RNA coming from feces just because of the nature of the sample. You know, everything in feces is degraded. So my, my advice will be just really be cautious when you're extracting those samples, whether you're doing, especially if you're doing manually, because if you're doing manually and you're opening those tubes every time, you know, and you can cross contaminate between other samples and especially doing the spin down when you have to flush down the flow through. That's where I was more concerned because if you're not doing it properly, then you're contaminating, contaminating on your samples. So the reason why I love the Maxwell so much was because it was way more hands off time. You know, I would do beat beating pre processing then loading the instrument and be away with it. That really helps streamline my process because like I said, I don't know why. Where are the DNA whisperers out here? There's some people that are really good at it, and you give the same sample to someone else and then you just don't get the same you. And it's not the person. But I don't know what's in the process that we're doing differently. And Maxwell was able to get like good youth DNA from all of those samples. That really helped. As a follow up question mentioning beat beating, I, I'm curious to learn more about that protocol that you implemented just because I've, I've seen from, from publications just also in the community that potentially lower, you know, heavy lysis, whether it be through B beating or even enzymatic that that tends to cause additional shearing for DNA or RNA. Did you maybe experiment and and look to try different settings or optimizations or for that pre processing step? Yes, and I think we even have an app note on this, so I don't know if Sarah want to comment more and I can. Yeah. So we definitely have application notes about this. I guess one of the things that I would consider when you're thinking about doing your bead beating is what are you looking for. So really if you want to do those like total microbiome studies where you want to see everything that's in the sample, you want to make sure you're lysing your Gram positive and your Gram negative bacteria, You will need to do a more aggressive bead beating and that will impact the DNA and RNA integrity going in, coming out, I guess of the purification process. On the flip side though, if you are trying to purify a single bacterial species, maybe it's a gram negative species that tends to be easier to lice, maybe you don't need to be as aggressive with the lysis condition and you might be able to actually skip the beet beating process in general. So I think the biggest consideration is what exactly are you looking for? You're looking for everything that's there. Be aggressive. If you're looking for something and it's known to be fairly easy to break open so that you can expose the nucleic acid, you can use a gentler places. Protocol and it's OK to add to that. I think that's that's great that you mentioned this Sarah, because on my study specifically I was looking for micro RNA as to be used as biomarker, right. So I wasn't looking for any microbial communities. I was looking for the host microne. And why pieces, right. We chose pieces because it was easy sample time to receive from any lab, but also because we have shedding of intestinal tissue every day. So our hope was that instead of doing a biopsy, we could just measure in pieces what are the microne reflected in the tissue of those patients. So to Sarah's point, we weren't looking in that case for microbial community, you know, even though I do a lot of those, we were looking for a host micro name, so. And also thinking about looking at host DNA again, one of the things that one of the projects that I had done was looking at canine genotyping based on feces. And I found actually, you know, thinking about where you'll most likely find those intestinals shed cells, right, would be on the outside of the sample. So I found that it was really more effective to swab the outside of the sample versus taking a bulk fecal sample into the purification process for that specific application. Oh, that is really interesting. I didn't know about that. That is also, well, the microbiomic kind of want to like, you know, get the whole picture. But for you, it was helpful to just get the outside because you were looking for those shedding cells. Yep, Yep. Question just came in through the chat and for for Pat and Sarah and it's kind of along the lines of is bead beating even necessary especially with your experience for fecal samples, Do you think that that's required? Is there an approach where people are using more enzymatic base and chemical digestion versus some of the mechanical and more physical? Curious to hear your thoughts. I feel like if you're doing microbiome work, trying to understand microbial communities, you absolutely have to do big beating. There's no way around it. I mean, I couldn't find a better method to just really get the data. And I think there's some publications out there showing you the difference between a sequencing run if you do be beating and if you don't. And it's huge. So if you if that's the goal, which is looking at microbial communities, I would definitely encourage you to do be beating for like I was just pointing out that maybe for 9 doing more host then we may not need that super high disruption. I just don't know if I would completely take the beating out. What are your thoughts, Sarah? Would you? Actually, so for the canine genotyping work that I did, I did not do any bead beading. So essentially I wanted to be as gentle with the lysis as possible because we know that mammalian cells are fairly easy to lice. So I was just relying on the strength of our lysis buffer to lice it versus using anything kind of mechanical or any additional enzymatic lysis protocol because I was specifically interested in looking at the host DNA and didn't wanted to minimize the amount of any of that microbial DNA in the application of canine genotyping based on feces. Pat, we took a look really a lot on the methodology for for success of a sample purification. One thing I'm curious to learn more are the tools that you employed to really look at the quality of RNA. You mentioned tape station. Are there any other maybe spiking controls, things that you utilize along the way that really look to evaluate the the overall workflow? Yeah, and this is very tricky and I love that question because I feel like if you ask majority of people what they do for quantification, they would just yell Nana drop and they'll just go off those values like they're the Bible, right? And it's really, we need to be really cautious when we look at Nana drop values because they are like I said before, like there's spectrophotometer methods and there's like fluorescent probe methods. And when we do then a drop, which is almost like a spectrophotometer, we are not only measuring DNA, we're measuring other impurities that may be on the sample, some protein contaminations and other things as well. We're using fluorescent probes, like using quantum fluoro on the Qantas. Then we're actually binding those probes to our DNA or RNA and measuring exactly the nucleic acid that we have. So it's not uncommon to see a very high nano drop rate for one sample be very low in quanti flora for the same sample just because in quanti flora we're seeing the true amount of nucleic acid in that sample. You'll be really tailored to what you're doing. If you're just doing amplification, then another drop should be enough so you know what's in there. But if you're doing is sequencing and spending way more money on then, then you definitely want to go, you know, buy analyzer, which is a tape station or like fluorescent probe. But I don't know, Sarah, if you have anything else to add. Yeah, I think you covered it fairly well there, especially when we're talking about a fecal sample type, there do tend no matter what purification chemistry used to be, some of those fecal components like humic acid that are that will come through with the final purification, those do absorb in the same range as DNA. And so that will throw off your nano drop reading. So anything where you need kind of a precisely quantitative sample going into it, for example, Pat mentioned sequencing, we would definitely want to choose a more DNA specific or RNA specific way of looking at the nucleic acid that you purified. So either using the fluorescent based dye or tape station would be good as well. As a follow up, two really good questions just came into the in the chat and it's more so along the lines of, OK, we talked a little bit about microbiome, but for blood what, what potentially might be some pitfalls, but also some suggestions to work with this sample type. And then as a, as a next stage of that, how can that what might be some criteria to look for when heading into sequencing? And are they the same versus PCR? Kind of interested in your thoughts. So for blood, I mean, a lot of it has to do with the number of actual like white blood cells that are in the blood sample. So we've seen some different behaviors of different animal blood. Pig blood in particular was one where we found that essentially there were a lot more white blood cells in the sample. And so if we loaded the same amount of pig blood as we would for a human blood purification, we were actually overloading the purification system. And overloading a sample into the purification can also cause low yield. So that's that that would be 1 outcome. So I think if you are struggling at all with low yields from blood, you would want to take a look at how much input or maybe try titrating the amount of blood going into your purification. And just speaking up on that, I think you mentioned something really good that not always more is better for when we talk about nucleic acid extractions, you know, I've some people see low yield and they just keep putting more sample expecting to get more. But like Sarah said, more samples can cause inhibition also. And so it's like it's a fine balance. So what is the optimal amount of samples for you to load on your extraction? So we have very good point. And then kind of the follow up to that, like the criteria for sequencing are there, you know, rules of thumb or markets that you look for to really ensure that when you head into a sequence sequencing run it will be successful? That's really tricky. I don't know, Sarah, if you have a magic recipe, I'd say it would really depend on what is the library and prep mat that you're using for the amount of DNA that you need. I cannot just tell you, oh, you need to use this amount. You really have to see. I don't know what method we're talking about if we're doing, you know, there's so many. So I would just say look into what is recommended for input. Yep, I, I would have the same recommendation. And I was just going to reiterate one of the points that Pat made during the talk, which was that even though the quality of her RNA looked low on tape station, she was still able to take that into sequencing and was able to actually interrogate different biomarkers. So I don't know that you can always count on the advice from. So if you were using like an alumina kit, for example, a certain panel of targets, they might have a recommendation for RIN RNA integrity or DIN DNA integrity scores and concentrations going in. But a lot of times that that's their way of asking, you know, trying to get the best quality data for you, but you still might be able to get enough data out even if the quality isn't as high as their method supposedly requires. Oh, great, great questions coming in just out of curiosity as well. And we, you know, we, we discussed a lot of the methodology, but just kind of expanded to potential applications that this technology methodology you think will really enable and support Sarah and Pat. You mean the automated nucleic acid extractions? Yeah, yeah. Like where do you see right now for vet Med a lot of resources being poured into in terms of applications at this moment. So Permega has a huge portfolio. Permega has over, I don't know, 6000 products. That's the last that I heard. All of them can be used into your entire workflow from the genomics bar, which is, you know, extraction of nucleic acid extractions or amplification of those reagents. But we also have a lot of cell based assays that you guys could use into your workflow depending on what you're studying. If you're doing a lot of cells, you can also use our viability. We also have Lumet for a Western in the well or Eliza in the well. So if those are the things that you're looking for in the vet Med, I would definitely ask you to reach out to us if we'll be able to provide your resources on what you could, how we could support you in your research. But as far as automating like nucleic acid extraction, I'd say my, when I was a grad student and I was looking into the system, I definitely thought that I didn't need it, you know, and I think that was the, the most common mistake because I'm like, no, it's OK. I only strike five samples at a time. I can always do columns and, you know, use the centrifuge. And for me the word automation was very prohibited because in my mind, people doing automation or people who are extracting 300 and 100 samples at a time. But I soon learned that that's not true. And Maxwell was really helpful because I could extract as low as like 1 sample or five samples at a time, optimize my time and get really reproducible data. And I would just say reiterate that a lot of the work that we are doing so the scientific applications group, we take existing Promega products and try to make them work for unique applications or different types of samples. So if if you do have a lot of that work is coming in to us through our interactions with customers. And so we really try to be responsive to what customers needs are. And so if you are interested in having us work with your particular sample type or you need some optimization advice, we're always happy to work with you on that. And again, reaching out to your sales representative to have them connect you to our group is always the best route out to get to working with us. Excellent, excellent. Well, really good, good discussions and and please for the audience if there are additional questions. This has been a really engaging, so thank you. Well, we appreciate everyone's time today for this webinar. And as I mentioned will be if there are any resources, please feel free in the console to download. This will be part of a upcoming educational webinar series. So the first one is really been focused on sample purification. But in the future, we'll be really looking to explore other areas, applications that we can look to support a lot of the veterinarian animal health space. So thank you. Thank you. Thank you. _1734933352022