Morning, good afternoon or good evening everyone. My name is Eric Vincent, a Senior Product Manager in the nucleic acid purification group here at Permega and I'd like to welcome you to our November forum. Simplify complex Nucleic acid extractions, learn how to build, walk away, automated protocols for FFPECCFDNA blood, etcetera. I'm going to start off by kind of going through a little bit of, excuse me, a little bit of housekeeping for you on how you can participate on the forum. You're in your browser right now and know that you can resize and move around the windows to to better see and participate in the forum. If you have questions for our panel, once we get to the panel, you can either add them in the attendee chat or if you would like to ask the question, you can raise your hand using a little hand raise button there and you can join on on camera and ask the question verbally. Whatever is your preference, you can give feedback in a survey. We're going to provide are, you know, continually to add ask questions or or add comments because we and we will have a short survey at the end of the forum just to gauge the usefulness for this year for for you because we obviously want to continually improve on what we offer to folks. So with that, I'm going to move forward and we're going to show a short video. All right, hopefully you found that short video. I'm a little educational. I realize there are a lot of folks that are looking to learn more about automation and it may seem a bit daunting thinking about all of the things to optimize, but like to let you know there are a lot of support available from from MEGA and also products as you think about automating extraction. If we think about Permega and what we can offer laboratories seeking to extract nucleic acids, we're uniquely positioned to provide extraction of the clay acid at all scales from manual column based methods to small medium scale automation, what we may call bench top automation up through high throughput or 96 well, nucleic acid extraction. Just to take a few moments to tell you a little bit about Permega solutions for our moderate throughput, we have a small automated platform that you may have heard of. It's called the Maxwell. We have both a clinical CSC or IBD instrument as well as a research use only or the RSC. And these are benchtop instruments. They can process moderate numbers of samples. We have a small instrument for one to 16 samples or a larger one that can process up to 48 samples simultaneously. These are characterized very very easy to use instrument. Each sample has reagents in a reagent cartridge that comes sealed with the reagents present. You peel the the sealant off of that, load your sample, perhaps do a simple pre processing with some samples, load onto the instrument, press go and all protocols take about 25 to 60 minutes on the instrument to process those samples. So this is kind of for for folks looking at moderate numbers of samples. You may one run, run one, you may run many or if you think about scaling up to larger throughput, Permega has the capability to assist in programming instruments for higher throughput including all of kind of the the large global automation companies including Hamilton, Tcan, Beckman, Thermo Fisher, Eppendorf and more. We have this as a service that we provide to customers who purchase our high throughput reagents. So there's typically if you're purchasing a large amount of reagents, instrument method development and training is actually part of the purchase price of the reagent reagents from Permega. And we have a global field of scientists that actually help to support those installations and trainings as well as troubleshooting as you optimize the process. When we implement high throughput workflows, it's really a three-step process. We kind of have a discussion to understand where is the laboratory, what are they seeking to automate, what equipment do they have. We might recommend slight changes. You know, as you noticed in that video, there are certain parameters that need to be met like a particular shakers or liquid handling to be able to do the process that you desire. And then we'll make a proposal, usually some sort of a statement of work to agree on the requirements to complete the the programming and installation and then install the methods on the instrument in your laboratory, train laboratory staff and of course ongoing service and support. So we help either we have existing methods, we can also optimize those for chemistries on your instruments, train your your staff, help people get comfortable running the instruments and taking care of them and then assistance in troubleshooting and improving efficiencies as you are using them. We also have reagents that are available in standardized kits. We also have customers that may want reagents provided in very specific manners. Maybe program may be set up so you have one bottle of reagent per instrument run or 1 bottle of reagent used per day. Or you want very large bulk supplies that you draw off of over a larger period of time. And, and we can do that through our custom process and we can really optimize that to fit hopefully your needs for your workflow as as well as your budget. So we have a lot of things that we can do to efficient, efficiently help you make things work well for your lab. And with that, I want to introduce our panel of experts for today. We have 3 scientists here, Doctor Doug Koresh, Rick Greigo, and Brandon Kruger. Doug is a Associate director in the Protein and Nucleic Acid Analysis Division of Research and Development at Permega. He focuses on the development of nucleic acid purification systems to yield high quality nucleic acid from challenging starting materials. They his team works on creating purification for all scales, manual and automated formats and also they they work on developing dyes from nucleic acid quantitation. Before joining Permega, Doug served as a Clinical Laboratory director at a molecular pathology company in Virginia, where his responsibilities including identifying, evaluating, validating, and integrating IVD and LDT assays within a clinical laboratory setting. He also managed BCL 3, BSL 3 facilities, and LED contract research on emerging and biodefense pathogens. He's an author of over 20 journal articles and several world and Italian patents, and he earned his PhD from the University of Wisconsin and complete his postdoctoral training at the Institute of Human Virology in Baltimore, MD. Rick Rick Greigville joined Permega in 2018. He is the supervisor of our Field Support Scientists for North America. He and his team support the implementation of Permega Chemistries on laboratory high throughput automation. Their support combines chemistry and instrument expertise to provide customers with tailored automation solutions, trainings, and support. This enables cut folks to implement our chemistries more rapidly without having to be a chemistry expert or an instrument expert yourself. Rick joined Permega after spending over 17 years with a molecular diagnostics company, 15 of those as a field automation and applications scientist. And during this time he helped over 100 laboratories automate their nucleic acid purification and molecular testing workflows on various platforms. He was also instrumental in successful development and launch of several Class 1 and Class 2, Class 3, excuse me medical devices. Rick has a also has a degree in chemistry from the University of Wisconsin, Madison. And last but not least, Brandon Krueger is a field support scientist with Permega Corporation. His main role is very similar to Rick's. He's utilizing the diversity of automated instrument platforms such as the ones I've mentioned, TK and Hamilton, Beckman and Eppendorf, Thermo Fisher, others to create and develop custom automated scripts using Permega reagents in customer laboratories or internally as a technical product expert for reagent and instrument applications. He joined Permega after spending thirteen years in a clinical research laboratory working with small molecule pharmaceuticals. During his time there, he led all automation out of opportunities on Hamilton robotics platforms as well as he serviced their Hamilton micro lab * platforms. So these are our our panel for today and I'm going to start with some of the questions that were asked as you registered. And if something else comes to mind or if you have a follow up question, please feel free to use the attendee chat and we will respond to those as we have time. So we, we've got several questions here. Some of these, a lot of people asked a very similar question. So your questions may not be specifically asked, but one very close to what you did. We're going to try to get those all in. So the first question is really a common one that we talked about and we addressed a little bit in the in the video. But Brandon, the first question was how do you prevent and monitor cross contamination when you're automating sample extractions? Yeah, Thank you Eric and, and and thanks for the the question. So first of all, monitoring for cross contamination, one of the things that we will typically do is, is recommend a checkerboard pattern of of male, female kind of across the 96 well plate. And then what you can do is you can use PCR detecting specifically for the Y chromosome just to confirm that that that Y chromosome has not been a cross contamination in and the areas where you would not expect it. So that's kind of a, a quick way to take a look at at cross contamination. As far as preventing and avoiding cross contamination, there's a wide range of of things that you can do with the instrument. First of all, a lot of these automated instruments have you know, filtered tips or unfiltered tips and the manufacturers don't necessarily care which you know which tips that you will use during the processing. But we actually prefer to use filtered tips to help prevent contamination of the instrument in general through through the use of the instrument over the course of using your samples. As we are optimizing a method, we will optimize liquid classes to help prevent formation of bubbles or droplets at the end of the tips, specifically when working directly with the sample itself. When you're working with clean sample, it's not as impactful, but we will still try and avoid those, those bubbles and droplets, you know, 'cause ideally you don't want those two to drop anywhere on the instrument as well. But monitoring for those, those droplets will help prevent, you know them dropping off at any point in time during the the extraction and that will help prevent cross contamination. There's another thing, a lot of times when you're using liquid handling automation, a lot of what I've been talking about thus far is, is more targeted towards liquid handling. One of the things that we try to do to reduce cost is, is reusing of tips throughout the process. And one of the things that we will do is we'll, most of the manufacturers at this point have some sort of tip isolation available in which you can utilize a plate or a chamber where when you drop those tips back off for a staging scenario before you go get another reagent after they've dealt with the sample. It basically puts each individual tip into somewhat of a chamber and isolates it from the other tips so that you do not have tip to tip cross contamination during the process. So there's a number of things that we can, we can really take a look at and on the liquid handling side, as far as particle mover instruments like our Maxwell or even if you go along the lines of, of a, a Kingfisher instrument, basically those particle movers are, are, are all built to prevent cross contamination. Specifically our Maxwell. There is no point where a, the plunger or, or the plastic that covers the magnet, there's no point where that plunger is going to be over the reagents or the processing area of a, a subsequent sample. They're all very linear working in the same space. So it'll help prevent cross contamination there that way as well. All right. Thank you, Brandon. Let's move on to the next question. Doug, hopefully you can handle this one. This question is there's a lab, a lab has had a project assigned where they will need to extract RNA from FFPE fixed tissues using a Maxwell kit. Therefore, we would like to understand a little bit more about the technique in the process before we start. Thanks for the question Eric and yes, I can answer this. There are a few considerations that you have to have when working with RNAFFPFFP is a very specific type of sample that is fixed. So you have to perform AD cross linking step along with a proteinace case step. And then you have to go through your very typical nucleic acid purification where you do all the things that were listed in the video such as binding a washing, eluding and so on and so forth. What we do with the Maxwell instrument is that we actually put together an entire package where you have all of the reagents and you, you have the instrument and you have the, the method very well optimized for, for purification in this case of RNAFFP. So the easiest way to look at this would be to request a demo of the instrument and of the chemistry and you would have everything in the box that you would need in order to actually run that chemistry. And then if need be eventually if if you purchase a Maxwell and you outgrow that Maxwell with throughput, then you would talk to people like Rick and Brandon to have this automated on a a larger instrument. Alright, thank you very much, Doug. Next question, kind of a more general question and we've covered a lot of this probably in the video. But Rick, someone's asking about kind of just in general tips for moving to automated DNA extraction. If you could kind of maybe summarize if you were a lab or someone assigned with starting to do this, what what might you be thinking and considering to be most effective moving forward to do that? Absolutely. So I guess one of the initial considerations you know, beyond kind of space facilities availability, budget availability is kind of the, the amount of complexity that a lab is, is looking to take on, right. So kind of the, the least complex automated platform would be like our, our Maxwell instrument where you can process, you know, 6 up to 16 samples or 48 samples at a time and their agents are all in cartridges and, and everything is, is really simple to use, really straightforward. Now some labs if, if you need higher throughputs, if you're willing to take on a little bit more complexity, you may be look looking at a plate based particle mover such as the, the Kingfisher platforms, whether it's a Flex or Apex, what have you. So there's a little more complexity there, but still, you know, they're, they're relatively straightforward. There's not a whole lot of of variables that are needed to be adjusted and in order to be successful. Now, if you need to go even beyond that and and say you're looking to do more than just magnetic particle based applications, you may consider moving into a a liquid handler now. When you're trying to automate the nucleic acid purification on liquid handler, there are certain devices that are critical. It was touched on, you know a shaker in the video depending on kind of the the plate density you're you're looking to utilize. Well, it's whether it's a A96 wall plate, 24 wall plate, 384 wall plate, it's important to get the an appropriate shaking orbit with your shaker. So most typically we process a 96 deep ball plates and, and for those we, we recommend a 3mm orbit shaker. If your purification requires a heated lysis or heated illusion, you'll also want to get a shaker that has heating capabilities and you'll want a an an adapter that mates well with your chosen deep ball plate just to have the most efficient heat transfer possible. In addition to you know, the heater shaker, you'll want to source it in an appropriate magnet. We are fans of the post style magnet. It it draws the beads off the bottom of the wells to the side of the wells. So we believe they provide for faster separations, more complete supernate removals and you lose fewer magnetic particles during the course of the waste removals. However, the one limitation is as far as illusion volumes. Typically you're limited to no smaller than around a 40 microlitre illusion volume. If you need to go below that, then certainly a ring style magnet may be more suitable for your application. And of course, aside from the liquid hand, they're moving liquid, it also need the capability of moving lab works because you'll need to move the your selected plate on or off your heater shaker, on or off the magnet during the course of the purification. So those are just some some things to keep in mind with respect to, you know, automating nucleic acid purification in your lab. OK, Thank you, Rick. The next question kind of goes back to our FFPE question from before, but now thinking a little bit more about going from something on a something a more of a fixed system like a Maxwell to moving to high throughput. So Brandon, I was wondering if you might be able to talk a little bit about some of the some of the more unique challenges with FFPE and how we might approach or overcome those when we automate in a 96 well format? Yes, certainly a few of those things have already been touched on with Doug talking about the complexities of the FFPE processing in general as well as Rick talking about, you know, depending on what your budget comes into. You know, what you have available for the purchasing of instruments, what you have available as far as space in your lab for instrumentation as well. What kind of, you know, what number of samples you're trying to extract over a, a period of time. And all of those kind of come into to to play across the board in, in terms of things you want to keep in mind when you're looking to make that transition to automation and, and really focus in on what your goals are, what's going to be most beneficial for you for the cost, you know, the budget that you have available to you and, and what cost you're willing to to spend. The biggest challenges are there, you know, are a few steps in that process, especially the D cross linking, which take a considerable amount of time that the majority of the time are are best suited as an off deck a segment of the method. Because you know, for RNA right now it's typically an hour de cross linking for DNA up to a four hour de cross linking. That's a lot of time for a large liquid handler, for example, to sit idle. So you know, but that doesn't mean that it can't happen. If you have the budget and want to keep it as hands off as possible, you can start integrating, you know, large devices and have an entire work cell where you know, you can have the samples themselves set into a rack that can be transferred to a, an air incubator as opposed to utilizing a, a shaker for for the incubation of those samples. So there's there's a lot of things to take it into consideration. Once you get past the de cross linking, then you're looking at just a, a normal extraction, any other extraction that you would typically be looking at. Once you get it beyond that, that lysis, that de cross linking and you have the nucleic acid exposed and you're moving into the binding stages, then you're just looking at the same thing as far as washes, you know, dry and then they looped. So there's a lot to think about on the scope of what HT means to you. And in the video it kind of referenced, you know, and Eric reference to this as well that, you know, we at per mega have this team of field sports scientists that are are here and available as a consultative role for you in the transition of your your per mega process into to high throughput. And and we're happy to be here as as a guide for you in that transition. Thanks Brandon. I I actually had one other follow up question as I, as I was listening to you for FFP, how do you get the samples in the 96 well plate? Do do you put scrolls in the plate or do you do something some other way to get those into the 96 well plate? Just thinking about you know that the scrolls of the FFPE. Yeah. And that's, that's one of the challenges. And as as we've been working with with customers, it's the getting the curls into a 96 well plate is challenging. Getting curls into a 1.5 mil tube, which have, you know, a much more substantial diameter to them is challenging because there's, there tends to be a lot of static creation in creation of the curl and the interaction with plastic creates a very challenging environment in doing so. And, and in, in my experiences working with FFPE, the majority of customers are not looking to put it into a 96 well plate because there's too much potential in cross contamination or, you know, partial part parts of the curl, you know, potentially flaking off into another well because it is such a fine diameter that you're trying to get those curls in. So a lot of people still stick with 1.5 mil tubes and that that's where it also becomes a a bit of a challenge in automating that because it's not a 1.5 mil tube, doesn't have a, a nice easy to use rack on a lot of these liquid handlers. So that's why some of those early stages tend to be a a little bit more manual at this point in time. We can set up the instruments to to add your, your protease, your lysis buffer, you know, whatever solvent may be used to remove the paraffinization. All of those things we can set up to add on the liquid handler. But a lot of times once you get to that stage, the lysis and de cross linking will will usually take place off deck. Now with that, you know, there, there are a variety of of ways. Not everybody is necessarily looking at a curl. Sometimes people are are looking at FFPE very targeted sections off of a a curl that is on a slide. So a lot of times they'll they'll scrape a specific portion of of some target cells that they're looking for and getting those into solution is, is a little bit easier depending on what you're using to scrape the slide. So transitioning that into a 96 well plate might be a little bit easier process depending on what you're scraping the slide with and the and the size of the instrument. Alright. And then Eric, if, if I may just add on a little bit more to what Brandon was saying that Rick and Brandon and the entire team, they actually work with us and with our scientific applications team. So obviously we have a lot of insight into the chemistry, what we've launched and what some of the other solutions that are out there. So we have a lot of experience through all of these, whether it's a Promega based DE paraffinization and transfer sort of workflow or if it's something else. But we've worked with several of these other groups in the past. OK. No, thank you for that. Next question, I'm Rick Berhard is asking about really thinking about establishing nucleic acid isolation on a TCAN specifically and they want to find a universal chemistry for different sample types. They didn't say whether it's DNA or RNA, but do you have some thoughts you can offer on that? Yes, absolutely. So I'm going to tailor this well, I guess initially to to DNA. So we do have a Maxwell HT genomic DNA kit. It's a pan sample kit. So we've we've implemented it at customer sites for blow it, blood, bone marrow, Buffy coat cells, tissue, I mean, you name it. We can even in many cases, you know, set it up to Co process multiple sample types. You know some labs there they're Co processing, you know, blood, bone marrow and cells, you know all within the same batch. If you have some of the more challenging sample types like such as tissue, if you were trying to Co process them, you you may need some upfront pre processing as far as the the tissue homogenization, you know, be before you'd be able to put it on to the instrument. So that's that's a great chemistry to use for DNA. Now shifting to RNA, we do have the Maxwell HT simply RNA chemistry and that chemistry, again, we've similarly to our genomic DNA chemistry, we've implemented it, you know, multiple labs, multiple sample types, whether that's blood cells, tissue, what have you. It, it works really well with, with many, many sample types and, and both of these actually scale really well. I think I mentioned earlier, you know, we, we typically process in 96 deep wall plates, but if you needed to process say larger volumes in that, you know, we could scale up to 24 deep wall plates. We've even gone higher than that. You know, we, we have a device called the HSM which stands for heater shaker magnet. In that device you could process up to 10 mils of blood, you could process up to a gram of tissue and even we can go the other way. So if you have a small input, say 10 to 20,000 cells, we could even process in 384 kind of the half deep plates. So both these chemistries work really well, you know, for it with many sample types and they scale very readily as well. And I would just add, you know, offline if if you have specific sample types of interest, let us know. Also, I believe in the resources at the end of the webinar, we do have a really large series of application notes where often times in response to inquiries by scientists for something that we may not have developed specifically the chemistry for, we will test and see how we can adopt our chemistries to those sample types. And we have a pretty high success rate and most likely we've probably tried what you're, you're interested in, but please reach out on that. The next question is about illusion volumes and, and someone asking you how, how can I, how can I approach or how can I aim for low illusion volumes for DNA extraction? Brandon. Yeah, certainly this is one that Rick already kind of touched on as well in talking about how we we really are big fans of post style magnets because it pulls the the resin to the side of the well and allows the the volume to settle in the bottom. Great. It works great, but it does create kind of a minimum illusion volume that you can work with. That's where the reference came in of potentially using a ring style magnet. In that instance, if you want to go lower than say 40 microliters, the biggest challenge can be is having a an entire setup that ensures that the volume of resin that you're using is going to be completely re suspended in the illusion volume. So regarding reducing illusion volume, you also need to keep in mind how much resin you are using. So when resin is provided to you, it is at a pretty high concentration and you know, for many of our nucleic acid extractions, we we recommend around 30 to 35 microliter volume of that highly concentrated resin starting to dip below that as far as illusion volume can create issues. But that's also where our experience with the chemistry and having great relationships with our various teams here at per mega, including the R&D teams that developed the chemistries. We can go back and, and really start to explore the possibility of, you know, what is the, what is the target reason that you want to reduce that illusion volume. If it is because you have a sample that is extremely low in concentration and you're trying to amplify the concentration of your final eluet, we might be able to work the the total volume of beads that we are putting into the chemistry. We might be able to reduce those because as you start to use a low a low elution volume with a high volume of resin, you're going to start getting bead carry over in that final elluot. Even if you use a ring magnet. And there was kind of a, a representation of that in the video where you know it there was just so much resin mass and so little illusion volume that you could see some of those those that loss in beads. So there's always a balance within the chemistry on what you want to accomplish when reducing illusion volumes or or really just trying to get a more concentrated final output. OK, thank you and and again. Usually the answer is it depends. So if you have a specific sample type system that you're interested in approaching with that, that's, that's again something that our field support scientists can either help program if you don't have the ability in your lab to do that yourself, or consult and offer ideas if you prefer to program the instruments yourself. So, you know, please use those resources, you know, both here asking the questions and also reaching out outside of the webinar format. Doug, I, I've got a next question is how can I ensure optimal license efficiency? It's a wide open question because they didn't say a sample type, but maybe you could offer a couple kind of general ideas on that. Well, so first of all, the good news is that with a lot of our, with all of our Maxwell kits and all of our Maxwell HT work flows on larger automation, we've actually done a lot of that work for you to ensure that you're getting optimal lysis. So one of the things that we do as a part of development is we have a series of different formats license buffers. We, we literally have, we have hundreds of different potential license buffers that we can use. And often times when we're early on in development of a chemistry, we'll, we'll start taking some of these different pieces and mix and match them to come up with the optimal workflow that fits both for the, the, the matrix, the, the, the sample type that you're working with, as well as with the downstream assay. So we, we really do go through and do that optimization for you up front. Of course, if there are specific examples where there's some question as to whether it's the best, the best workflow for you. If you have a different a different sample matrix that we have less experience with, then we would bring that within R&D and within our scientific applications to help ensure that you're getting optimal license. So the short answer is we do that work for you. The longer answer is that we can collaborate with folks and understand exactly what they're trying to do as far as the sample matrix and then for that downstream assay. And we we typically match up all of our entire workflow to suit that in the best way. OK. Thank you very much. Believe it or not, we've actually reached the end of the hour and I want to be respectful of people's time. There were questions that were asked that we did not get to. And for those of you that asked a question that we did not cover, we will try to reach out to you in the next couple days to a week to, to answer those questions. And if there's something that you wished you would have asked, please feel free to reach out to us. Also, take a look at the the resources from the webinar. There's a lot of additional information about answer to some of these questions as well as other resources on for mega.com. With that, I want to thank you all for joining. Please take a moment to fill out the survey at the end because it does help us as we plan for future content for these these webinars and forums. So, you know, if there's something that you would love for us to cover our, you know, to focus a question and answer session on something else, please let us know. And with that, I'd like to thank Doug, Rick and Brandon, and I would like to thank all of you for spending some time with us today. So thank you very much and have a great day. Thank you. Thank you. _1734783275185