Hello and good day everyone. My name is Monica Yu and I'd like to and I'm a product Manager at Permega Corporation and I'd like to welcome you to our webinar today which is improving SFPE purification workflows with chemical catalysts. All right, so before we move on, I'm what like to do a little bit of housekeeping on your screen. There are multiple windows and all of these are movable and resizable. So feel free to move them around to get the most use out of your desktop space. In this webinar. We're we have many ways during the webinar and we will try to answer these either in the chat or during the live Q&A session following the presentation. Now we there is a resource library window as well and this has a list of helpful materials in it. So you can, and this also includes a copy of today's presentation slides. Feel free to download any of the resources or bookmark any of the links that you find useful. After the presentation, we will also have a survey. Please take a moment to answer these these few questions. We do really appreciate having your feedback. And then you're also welcome to share this webinar with your colleagues so they can learn more as well. All right, so now that we've gotten the basic housekeeping done, let's go ahead and introduce our presenters. So today's presentation will be presented by Doctor Kevin Mayer. So Kevin is a Senior research scientist in the Protein and Nucleic Acid Group at Permega Corporation. He has a diverse background spanning molecular pathology, genetics, and biotechnology. He began his career as a research scientist in Mayo Clinic's Division of Experimental Pathology and Laboratory Medicine, where he studied HPV viral integration events in head and neck cancers. While at Mayo, he was he also interned as a laboratory technician in the Molecular Hematopatho Hematopathology group, excuse me at the Mayo Medical Labs. Before joining Permega, Kevin received his PhD in genetics from the University of Wisconsin, Madison where he studied genetic pathways underlying plant flowering time. In addition to these activities, Kevin has provided consulting services with the nonprofit by We Solve to assist Madison area biotech businesses. So once our presentation from Kevin concludes, we'll have a live Q and a session led by our scientific expert, Doctor Douglas Porsche. So Douglas is an Associate Director in the Protein and Nucleic Acid Analysis Division of Research and Development at Permega Corporation. He focuses on the development of nucleic acid purification systems designed to yield high quality nucleic acids from challenging starting materials. His team works on creating purification chemistries for both manual and automated formats and develops fluorescent dyes for nucleic acid quantitation. Before joining Permega, Douglas served as a clinical laboratory director at a molecular pathology company in Virginia. His responsibilities included identifying, evaluating, validating, and integrating IVD and LDT assays within a clinical laboratory setting. He also managed the SL3 facilities and LED contract research on emerging and biodefense pathogens. Douglas is the author of 20 journal articles and holds several world and Italian patents. He earned his PhD from the University of Wisconsin, Madison in 2001 and completed his postdoctoral training at the Institute of Human Virology in Baltimore, MD. All right, so now that we have finished introducing our two presenters, we are excited to dive into today's presentation. So with that, I will let Kevin take it from here. All right, thanks for that introduction. My name is Kevin. I'm a senior research scientist here at Permega Corporation and welcome everyone to today's webinar entitled Improving FFPEDNA Purification Workflows with Chemical Catalysts. So in today's webinar, I'm going to give a brief background on nucleic acid purification technologies. I will then discuss considerations of working with FFPE as a sample type and some of the unique challenges that is involved with working with FFE. So I will then go on to share some R&D work we've done here at Per Mega in which we've developed novelty cross linking chemistries for use with FFE. And then I'm going to introduce the Maxwell RC Accelerate DNAFFPE kit, which uses these chemistries and we will explore downstream applications using this kit including NGS. So our group is the nucleic acid purification group here at Permega R&D and our overarching goals are to develop solutions to purify any nucleic acid from any sample type for any downstream application. So did during development of our technologies. We must carefully consider what the target analyte is to be purified. We must understand our sample type deeply and we need to understand how this analyte is going to be used in a downstream technology. So today we will be discussing the purification of genomic DNA from human FFPE samples for a variety of genetics and genomics applications. So a nucleic acid purification can be broken down into two basic steps. The 1st is sample pre processing, which involves lysing cells to release nucleic acid and the second is purification, which isolates those nucleic acids from all other components of the sample. So depending on the sample, we employ a variety of pre processing methods. In particular, for FPE we use detergents, some extreme temperature incubations, and a variety of enzymes including proteases and nucleases. So once the nucleic acid has been released and cell and tissue debris is clear, we can begin purification. This can be done in a variety of ways, but examples here is Permega's line of Maxwell instrumentation, which carries out automated purification using a cartridge system. So a lysate is moved into the well of this cartridge and using paramagnetic resin and movement, the system can bind the nucleic acid, wash away impurities and dilute high quality DNA. So today I'm going to share work we've done to really improve the preprocessing of FFPE samples. And this preprocessing maintains the same downstream tried and true purification platforms that we have. So let's take a closer look at what is involved with the preprocessing of FFPE samples. So FFPE is embedded in paraffin. And before we can lice the cells, we need to remove the paraffin. So typically with either mineral oils or xylene, you can remove this paraffin and separate the tissue. And then there's the addition of a lysis buffer. So this typically contains a buffer and a detergent. So this rehydrates the tissue and it license the cells to release DNA. There's been a protease digestion, so this further promotes Isis of cells and it helps to solubilize cross-linked DNA and protein complexes putting in them into solution. And then there's the important step we call the D cross linking reaction. So this is typically done at high temperatures for long times. And the goal of this step is to reverse the DNADNA and DNA protein formal and cross links that are in the FFP tissue. Finally, there's a nucleus treatment to rid the lysate of RNA and then you can purify it on your downstream platform. So today's talk is really going to be focused on innovating chemistries for the D cross linking reaction. And before we dive into that, we really need to look and examine FFPE and how these cross links are formed and why they're formed. So what is FFPE and how is it used? FFPE stands for Formal and Fixed Paraffin Embedded Tissues, and it is a really important clinical sample type that contains a great deal of molecular information about patients and their disease. So FFPE is typically made from surgically removed tissues, and historically morphological analysis was used to characterize and study these samples. However, in the past couple of decades, solutions for purifying macromolecules such as nucleic acids have had great progress and now a gamut of molecular biology techniques can be used to study FFP samples for a variety of of applications, diagnostics, disease, R&D, drug development and biomarker discovery. So there are some unique challenges to working with FFP as a sample type. And the first is that FFP is a highly variable sample. There are both biological and technical sources of this variation. So working with any human tissue, you're going to be up against biological variation including the age and body composition of the patient and which the sample was procured from. And you have to consider the metabolic and disease state of that tissue. And finally, there are impurities that might be inherent to the tissue, such as urea from kidneys or digestive enzymes from the pancreas. But there can also be environmental impurities that accumulate over the life of a patient, such as tar and a smoker's lung. So beyond this biological variation, FFP is unique. And that it is undergoes a very rigorous process to create this and this introduces technical variation. So this is a fantastic review paper that examines all of the pre analytical factors that could potentially affect quality of nucleic acids purified from FFPE. So from before the sample was put into a formal and buffered solution, the formal and fixation process itself and the storage of the FFPE sample can potentially impact yields and quality of nucleic acid from FFPE. So the second challenge working with FFPE is that nucleic acid from it is often poor quality. So if we consider DNA purified from fresh or frozen genomic DNA, it's high molecular weight and the sequence is intact, but FFPEDNA is typically quite sheared. There's cross links on the nucleic acid and there can be other damages associated with it. So certainly the sample preparation itself can introduce these issues. So the formalin fixation process itself, it's the basis for what is preserving the tissue. These cross links can interfere with downstream assays and how you use the DNA and there's evidence that this process is also shearing and contributing to degradation of of the nucleic acid. And through storage as well, you can create oxidation events that can be problematic as well for applications that use ends of DNA. More recently we've been learning that the extraction process itself, particularly the D cross linking step in the pre processing can contribute to poor quality. So double stranded DNA breaks are are common. Other breakages such as overhangs, NICs and AP sites can be problematic and there can also be base transition. So these are sequence artifacts typically through deamination, which we'll talk about later in the webinar. So before we talk about reversing these, let's look at how these cross links form in cells. So formaldehyde is a is a very small molecule. It's extremely electrophilic and it is cell mem permeable and nuclear membrane permeable reacts with amines that happen to be on proteins or RNA or DNA. And formaldehyde happens to be a bi functional cross linker and that it can react with amines twice and this forms the basis for how it forms cross links. So here we have an example of two proteins that are cross-linked by for a formaldehyde molecule to DNA molecules cross-linked and a DNA protein cross link. So different molecules undergo different levels of fixation and DNA is heavily cross-linked and an FFPE tissue. And the reason for this we can look inside the nucleus. DNA is found in complex as a nucleosome. SO150 base pairs of DNA wrap around a an octomer of histone protein and these histone proteins are very rich and amine containing amino acids such as lysine and arcanine. So if you consider formaldehyde, it's the BI functional cross linker. It it really is only cross linking molecules that are in close proximity. Well, DNA is very tightly associated with the histone and with the richness of amines and these two molecules, formaldehyde finds its way into the interface and can create cross links between the DNA and the histone. So if you consider there's 30 million nucleosomes per cell and there are dozens of available amines to be reacted with, this is a lot of substrate and this is the basis for why DNA is so heavily cross-linked and FFPE tissue. So if we look at a chemical reaction mechanism of what's happening, I'm, I'm showing here a lysine residue of a histone and a guanine base of DNA. So empirically this appears to be a the most abundant DNA protein cross link and formaldehyde is being extremely electrophilic will undergo nucleophilic substitution of this amine here in the in lysine residue and this will form a methylol intermediate which goes on to dehydrate to form a shifts base intermediate. So this shift base is still electrophilic, it's weaker than free from aldehyde, but this will attack approximable amine like that of the amine on a guanine base. And we have our kind of our canonical DNA protein cross link. So it's important to note that each step of this reaction exists in an equilibrium, but the stable crossing products are are most strongly favored. And because it's in an equilibrium, it can be reversed, and enthalpy, either using pressure or more commonly heat, can promote the reversal of these cross links. So why is the D cross linking step so important for FFPEDNA yields and quality? So to demonstrate that, we have an experiment here and we'll just kind of walk through this slowly. There's a lot going on. So we took FFPE cell lysates and subjected them to increasing D cross linking times at 80°C. We then purified the DNA with a Promega Maxwell RSC kit and then we assess the DNA yields in two different assays. The first is with a Multiplex PCR assay and the second is with capillary electrophoresis. So let's first take a look at the DNA yields as assessed by the PCR reaction. So first to note is that this assay can amplify 3 different targets of different sizes 75150 and 300 base pairs. And you note that the 75 fragment is more concentrated than the 150 or 300. And this is because DNA is typically fragmented in FFB. So at short D cross linking times of 15 minutes to an hour, we get very little amplification. And this is because the DNA is still cross-linked. It doesn't denature well. The DNA polymerase enzyme has a hard time amplifying and it's not till 4 to 8 hours do we reach our peak optimal de cross linking time for amplifiability. And past that you can go too far and we start to see a drop off in amplifiability. So the DNA quality is being compromised here and probably further degrading. And so we can look at how the DNA purified from these migrates on, on, on gels. And we can see early on there's very little soluble DNA, but over time there is what appears to be a high molecular weight band. But it's important to note that this DNA is an amplifying well. So it's still cross-linked and migrates high by virtue of being cross-linked. So it's not until after 4 to 8 hours do we start to see it migrate to its true size and which it has the optimal amplifiability in, in PCR. And then past that point consistent with our our PCR data, it starts to degrade further. So the take away from this slide is that every FFPE sample has an optimal D cross linking time. Too little D cross linking and the DNA might appear high molecular weight in certain assays, but it's still cross-linked and it's not going to work well in a lot of down streams that use enzymes or denaturation and hybridization. On the flip side, too much de cross linking and you can start to degrade your DNA and compromise its quality. So there's certainly an optimal here and we seek that optimal in our purification systems, so. As I had mentioned, the industry standard for this currently is a long incubation time at high heat. And so at per mega, we initiated a project to find alternative means through using chemical catalysts to promote and selectively reverse these cross links that are found in FFP. So here I'm showing results from chemical screening that we did in collaboration with Permega's chemistry group. And so there's quite a bit here and we'll walk through it slowly, but essentially what we're looking at, the colors here represent relative amplifiable DNA yields as determined by APCR assay. So each column here is a different experiment with a different chemical compound that are numbered numerically. And we've also ran a couple controls alongside each experiment, a mock for our D cross linking. So this is an optimal D cross linking time and a 30 minute D cross linking. So this is suboptimal. And if you look at the difference between yields here like say at #15, you can see there's you're getting about one to 10% of amplifiable DNA when you D cross link at 30 minutes. So we introduced these compounds right before the D cross linking reaction into the lysate at 20 or 100 millimolar and at various starting lysate PHS. And for example here at 15, the 100 millimolar PH74, we saw a bump and amplifiable yields about 20% relative to the mock 30 minute control. So we took this chemistry compound and working with our chemistry group, we explored the chemical space around it doing structural activity relation and we've iterated the screen and we were able to enrich for compounds shown here that appear to be catalyzing the reversal of these cross links. So in other words, putting these in at their optimal concentrations and pH gives just as much DNA at 30 minutes as our optimal previously identified optimal for our control. So we next went through to kind of winnow down this these collection of catalysts, we had to find those that had desirable physical properties, but then we tested them in real human donated liver FFPE tissue. So we typically do several purification replicates and our development and this is to overcome intro any variation that you might see within a sample and we would run several different controls, a four hour D cross linking that's the optimal a 30 minute and then our catalysts. So you can see here the same Multiplex PCR assay that I introduced previously. We have quite good yields here from this liver sample with a protocol that is using a four hour D cross linking. Where's the 30 minutes gets considerably less amplifiable DNA and two different compounds here one and two can provide just as much of that amplifiable DNA at the short 30 minute de cross linking times. So we've also used other assays in development including dye biting assays. So shown here is a Permega's Quanta floor system, which is similar to a Qubit system that it uses a double stranded DNA dye and fluorescence to detect quantify values of DNA in solution. And so we're able to detect good quantities of of DNA in these solutions. And this DNA purified from these catalysts is also able to absorb UV in an unexpected way. And so here we're showing measurements of UV absorbance taken by nano drop instrument. So finally seeing how this how these purified DNA electrophoresis on on capillaries, they electrophoresis to similar sizes as a four hour optimal optimal D cross linking protocol and they're picking up the dyes that are embedded in these capillaries without problem. OK, So what we've what we've finally come to is a formulation of a solution that we're calling the accelerate buffer one XP 1. So this contains the cross linking catalysts. It contains a proton donator, which is an acid and a buffer and a yellow indicator die so that you know when you put it into your lysate. And what XP one is essentially doing is promoting a mean formation and transmination of a shift space. So this is effectively catalyzing the reversal of these formal and cross links and mono adducts that are on FFPEDN A. So we've taken this solution and created a kit called the Maxwell RC accelerate DNAFFPE kit. And So what this kit offers is basically a much faster pre processing than any existing kit and pairing this with the high performing Maxwell cartridge purification chemistry creates a very streamlined and fast and easy protocol to use. And so there are other benefits that we've encountered with with this kit including better NGS performance in certain metrics, which we will go into here shortly. So taking a look at the time aspect of the kit here we have preprocessing times, protease and D cross linking incubation times and temperatures for a variety of kits that are available. So you can see that in Gray here. Typically there's a long protease digestion and a long D cross linking digestion can be several hours to overnight. So certainly existing Promega kits, you might be familiar with the RSCDNA or the F of PE plus are are are competitive with that. But what we're hoping with the Accelerate kit is this ability to do this in a much shorter and gentler way. So it's providing a shorter DE cross linking time, necessary DE cross linking time and it's using lower incubation temperatures. OK, so let's take a look at the workflow itself. So to your FFPE sample you will add mineral oil, give a brief vortex and then deep paraphenize at ADC for two minutes. You will then add a lysis buffer master mix, which contains detergent, a buffer, a protein Ace K and a blue dye, and this helps lice the cells. And then there's a distinct protein Ace K digestion. So this proceeds at 56 Celsius for 15 minutes. The Accelerate Buffer XP-1 solution is added and the cross linking occurs for 30 minutes at 80°C. You then go through your RNA, say treatment and then purify the DNA on the Maxwell RSC. So the Accelerate kit is compatible with all tested QC and downstream assays. And I do want to highlight that most of the development of the kit was done using a quantitative PCR assay. And the reason for this is that a PCR assay is a great indicator for how well your DNA is going to be used in most downstreams. It denatures and hybridizes DNA and it uses an enzyme to essentially give a readout of the DNA. Now other assays like absorbents, fluorescent dyes, or gel capillary electrophoresis are you typically very informative for highly pure high quality nucleic acid before FFPEDNA. It's more of a complicated readout because these assays, in particular UV absorbance can always tell if the DNA is still cross-linked or not. So well, these assays are cost efficient, they're fast. They do typically give you less information and are less predictive of success for your downstream. So just keep that in mind. But we will go through performance of the Accelerate kit with all of these assays. OK. So first we tested the performance of the Accelerate kit and 13 different human FPE tissues. So for each tissue we used one to three donors and typically we like to do 2 to four extractions per donor to get across that variation that we sometimes see. So we compare this to a protocol that uses a four hour de cross linking and we used several assays. So we used a Multiplex PCR assay, a dye binding assay, the quanta floor system and then UV absorbance with nano drop. And what we can see is kind of a typical spread of concentrations depending on the sample and the tissue type. But we see very good performance of in terms of yields no matter how you measure the DNA from all three of these assays. So an important feature of Adna purification kit from FFPE is that it can scale with tissue input. So what we mean by that is using either small amounts of cells or large amounts of of cells or tissue. And so this is particularly important for FFPE. So if you're trying to macro dissect some tumor cells in your section or if you have a small surgical biopsy, you'll need to be able to purify DNA from these small precious amounts of cells. On the other hand, if you have increased inputs, so if you're using an NGS assay that requires a high nucleic acid concentration or if you're working with low cell density tissues such as breast tissue, you're going to want to put in more sections. So the Accelerate kit performs very well with different input volumes. So here we've kind of simulated small amounts of of input by taking a half quarter and 8th sections and we see really good linear yields all the way down to a single digit nanogram per micro liter concentrations. And conversely, we can scale up. So we're taking multiple sections here and we can reliably get yields up to hundreds of nanograms per micro liter. OK. So the we also evaluated the performance of the Accelerate kit in a digital PCR system. So here we purified DNA from: and breast FFPE using the Accelerate DNA kit or a 30 minute D cross linking mock or a four hour D cross linking protocol. And then we used bio rad reagents and instrumentation to do DDPCR. So you can see here in this in this kind of this middle panel that DNA that's not sufficiently the cross-linked has a hard time partitioning. And of course there's just less amplifiable droplets and the Accelerate kit has great partitioning and really good performance in this system. OK, throughout development we have been conducting next generation sequencing to intimately understand the quality of DNA that are coming from our purification system. So the this is a busy slide, but the important, the important information to take away is that we use a targeted NGS amplification based library. So this is aluminous ample seq for cancer hotspot panel. It amplifies 207 different amplicons and these are kind of spread out across different hotspot genes, CNBS and fusion drivers. We've also included a couple DNA controls here. So the first is G3O4, which is a high quality genomic DNA, so it's purified from fresh tissue and HD8O3, which is a Multiplex reference control that is formal and damaged and simulates formal and damaged genomic DNA. So we took that alongside DNA purified from the Accelerate kit or a four hour de cross linking and made libraries. We did library QC, we sequenced with a an aluminum I seek instrument and then we did S&P analysis with within the Lumina space space environment. OK. So walking through kind of the different results that we have, we are able to make expected size and high amounts of of library with the accelerate workflow shown here. And this is comparable to libraries that we can get from high quality genomic DNA that you can see here in this lane. So if we contrast that to DNA that's still has been formal and damaged and that is still cross-linked, you can see HD8O3 here really suffers. And so we get expected library yields at the expected quantities. So looking at the quality of reads that we get from a sequencing run, we have a high percentage of reads that pass filter that are aligned to the reference and that are on target, similar to high quality genomic DNA. And when we do a SNP analysis, we can reliably detect germline mutations and somatic mutations down to the limit of detection of this assay. So those doing genomics with FFPE may know that NGS can be challenging. In particular there can be high levels of sequence artifacts in FFPE and a common sequence artifact is AC to T snip. So this is a result of cytosines 4th position amine spontaneously deemanating and this changes the cytosine nucleotide identity to a year soul which through PCR will base pair with adenine and then threw a subsequent round of PCR base pair with thymine which results in the C to T snip. So this is a well reported phenomena certainly in the literature, but the cause of this is not exactly known. So we have evidence that the de cross linking reaction is in part driving C to T rates. So here we can see C to T rates as a function of the cross linking time. And you can see that the cross linking incubation is actually driving these S and PS and interestingly, past a certain point they disappear and this is probably due to the year sole nucleotide further decomposing to a species that can no longer be amplified and is therefore no longer represented in the library. So we have found that DNA purified with the Accelerate DNA kit tends to have lower C to T rates compared to protocols that use longer D cross linking. And we think this is in part due to the shorter D cross linking time that the kit can use. Now there are approaches to limit the impact of the C to T artifacts and one approach is the use of your soul DNA glycosylase. So UDG is an enzyme in the basic decision repair pathway and it cleaves the in glycosidic bond of your soul from DNA to leave an AP site. So this mechanism can be used to so-called repair the FFPEDNA and indeed UDG treatments do reliably reduce levels of some C to T artifacts. So here we have an experiment where we see high levels of C to T and a protocol that uses a four hour D cross link and the Accelerate which uses a shorter D cross link reduces the C to T rates. So here we have a competitor kit without the use of UDG. It has high levels of CDT and blue, but treatment with UDG lowers the C to T rates and it does so quite effectively. However, this is not a so-called repair kit because molecules of DNA targeted by the UDG are not amplified due to the AP side left by the cleaved uracil. So they do not get amplified in the library prep and they do not get sequenced and therefore aren't represented in your data set. So this ultimately has the consequence of reducing your library complexity, which is not desirable. There is a second limitation to the UDG approach and that is it can't remove all C to TS. So you can see here in this data set that there are still some orange dots here. These represent C to TS that are remaining after the UDG treatment. And this is because of deamination of methylated cytosine. So methylated cytosine otherwise known as DNA methylation is an epigenetic mark and it is found in CG context in the human genome. So if methylated cytosine undergoes deamination like shown here, this changes the nucleotide nucleotide identity to thymine, and thymine is not targeted by UDG. So the effect is that BC to TS will remain after UDG treatment, and that's exactly what we see. So the Accelerate kit can lower C to T snips that are found in CG contexts as shown here, while UDG treatment has no effect. So UDG treatments do reduce C to TS, but they cannot remove all C to TS, including. Those that are a result of DNA methylation being deaminated and they have the unfortunate consequence of reducing library complexity. So this is in contrast to the Accelerate Kit, which we think is just creating less CD TS in the 1st place because of this shorter D cross linking incubation. So with that, I'd like to summarize today's webinar. So we've talked about FFP as in a sample type, how extremely valuable of a it is in studying disease. But it's challenging to work with and that is because nucleic acids are extensively cross-linked and FFPE tissues. So the D cross linking incubation is necessary before the DNA can be used, but it does impact FFPEDNA yield and quality. So we've introduced the Maxwell RC Accelerate DNAFAPE kit. It's fast and easy to use. It employs a novelty cross linking catalyst chemistry that we've developed at per mega. And it has high compatibility with a variety of tissue types, a range of input volumes and really all the downstream assays that we think you're going to need to test. And interestingly, it does have improved certain NGS metrics like reducing levels of C to T snip artifacts. So with that, I want to say thank you for your attention and we're happy to take questions in the live Q&A session. Thanks. All right. Thank you for that, Kevin. So now we are moving on to our live Q&A session with Doctor Doug Porsche. We had a few questions in the chat and since a lot of these kind of came in closer to the end of the presentation, I figured it'd be easier to ask them directly to our expert. So the first question that came in was does the Accelerate Buffer XP-1 contain any dangerous reagents that would require pre processing to be done under a ventilator or in a fume hood? Now that's that's a great question. So there are no organic reagents as a part of this chemistry that would have to be processed under a fume hood. So you're safe to do all the pre processing directly on your bench. And then we had another question come in. So with the license buffer not the nature the protein ACE K? Or is there an equivalent to using the old incubation buffer to avoid this? Yes. So in in many ways it's very similar to the previous incubation buffer that we've used it. They're very highly tuned to get that core cooperative denaturation that you get by using some some chemical means as well as the the enzyme procase. So they're balanced in a way in which you actually get that that kind of event where they both work together in a in a cooperative way. Another question, can this kit can you use the rapid de cross linking with RNA extraction or maybe expanding on that proteins? So we've started to study that as well. We know that it will work with RNA. It's something that we're looking into further as well. As for proteins, we've seen that there's a huge need to get some sort of rapid de cross linking into the protein world as well. So we're certainly looking at our accelerate buffer as a potential for both RNA and for protein work in the future. Another question, is the protocol the same for low input tissues or high inputs? Another good question. We have looked at scaling the reagents and we found that we don't need to scale the reagents in the bounds of what Kevin has been describing going from a very, very small section to approximately 3 sections. It it all works quite well. So you use just the same protocol throughout whether you're using a very small quarter of a section or if you're using multiple sections in the same in the same pre processing. And I guess expanding on that, we had another question related to this. So does the amount of paraffin impact the isolation workflow when you're using it with the Accelerate kit? So it's a bit sort of around the same realm of the scaling, yeah. So we certainly looked at that with extensive testing including adding additional paraffin sections on top of just our normal tissue sections. Then we found that there was a very limited a fact of the paraffin. Obviously if you would overload the system significantly, you would start to run into issues with the mineral oil de paraffinization step. But that that is something that we've looked at and we do have a guidance on that if you start getting outside of what we would suggest. But right as of right now it works. It works great with up to three sections. So a question from someone who's familiar with some of our other Maxwell kits. Would you recommend preprocessing? If you're using the F of PE Plus kit, would you recommend using preprocessing with mineral oil as well? So the different workflows, they have their different advantages as far as workflow and as far as performance. And if you're happy with how that is currently going without the mineral oil, I would suggest to continue doing that. But this could absolutely be tested as well if that's something that would be more suited to the the workflow of the individual laboratory. Another question, is this accelerate buffer available only for the Maxwell workflows or do we also have a manual kit for this? It's another great question. It is something that we're looking at. We have worked with our scientific applications team to develop work flows in which you could incorporate that accelerate buffer and it's something that we're looking into further commercializing and and bringing to market. And I guess expanding on that one, similarly someone has asked is the accelerate buffer available stand alone? Currently it's not available as a stand alone, but it's something that we're we're looking to engage with with different collaborators and customers for other, other work flows or high throughput. And if that's something that is interesting, I would suggest to reach out to the local Promega Rep and and try to bring that back to R&D and we would we would love to work with you on that. All right. And then another question, have we tried using this kit with any forensic samples before? We, we really haven't up until this point, but it is something that we could certainly look into. Of course, per Mega does have a great history and working in the genetic identity and forensics field. So that's something that we would certainly leverage if there's a need for that, we would we would certainly look to incorporate this, this accelerate workflow for those samples as well. Kind of expanding on that theme of, you know, different applications, do we have any plans to test this with something like an Oxford nano nanopore flow cell? So we, we've done a significant amount of testing on Oxford with a lot of our other projects and it's, it's something that we can look at with, with this one as well. Typically we've, we've started with a focus on on some of the, the both the amplicon sequencing as well as the whole genome sequencing on a short read sequencing. But it is something that we're we're looking at on the Oxford as well. Another question, have we tested the accelerate buffer essentially this workflow with fish? So that's the fluorescence in C2 hybridization. We have. We have not up until this point, but it's certainly something that we could look at. A question on workflow. SO this person says that they routinely do FP cuttings during the day and then a sort of overnight digestion. Do you have any recommendations how you could maybe integrate this kit's workflow to fit that kind of timeline? We certainly could. Right now, our focus with the launch of this kit is to reduce that that timeline to make it so folks could do that in a single day. So you could accession the samples and you could, you could run your, your experiments and, and get it on to the, the, whether it's the sequencer or you're downstream later in the day. But it is something that we could certainly look at supporting, which would be most likely an overnight incubation at a much lower temperature, which would again help maintain that DNA quality. And I guess another similar question to that that came in was, So what happens if you use the accelerate buffer, but you extend the D cross thinking time to longer than what was what Kevin was suggesting? So we've, we've found that it's a very delicate balance of temperature and and of time that you have to typically reduce the temperature if you go longer because otherwise you'll start to fragment your nucleic acid. As as you recall in the presentation, Kevin showed kind of that balance between what you see in the tape station and what you see in the amplification. And there's kind of a a fine line balance of of where it it levels out at getting the maximal amplification, which is what was used to develop the timings for this kit. Another downstream question would would this kit extraction kit work upstream of spatial transcript, I'm sorry, spatial transcriptomics? I I think that it could. We haven't tested it up until now, but as I mentioned, we have looked at RNA and protein work as a part of some of our investigation and developing this workflow. So I would imagine that it could be integrated, but we haven't tested it up until this point. And then more of a general question, do you have any suggestions on the best way to quantitate F of PE DNA, DNA from F of PE samples? Yeah, as I mentioned, we we have done a lot of this work really based on quantitative PCR as well as by next Gen. sequencing because we feel that those are the most the the functional assays that most people are doing. So instead of looking at this from kind of a tape station or absorbance or or even fluorescent dye, we wanted to make sure that we were getting the most representative QC that we could come up with as far as ensuring that you would see success in the downstream assays as well. And. I think so. Another question sort of following up on a previous question. So you mentioned compatibly with RNA, has compatibility with reverse transcriptase qPCR been demonstrated? So we, we have done RTQPCR in looking at at at suitability for RNA workflows and we found that we do not have RT inhibitors as a part of this process. So it does work quite well with reverse transcriptase. Question. Do you have experience in further de cross linking extracted DN as? Typically we we typically will do the pre processing and run it through that way. So we have not done studies looking at de cross linking of of extracted DN as I would imagine that this sort of approach would be helpful because you get some more of a gentle de cross linking. You could probably look at much shorter times if you have a relatively pure DNA that's just carrying over some some cross link materials. But we haven't tested it tested that up to this point. OK, a lot of great questions coming in. Thank you everyone. I think though, we finally caught them all and so they came in fast and furious just doing Crystal. All right, OK, I think if there are no more questions, thank you very much, Doug for that. And let's All right. So if you do come up with other questions later on, please feel free to reach out to us again. So you can reach us at techserv@promega.com and also check out our website to see other interesting webinars that are coming up. _1727885970202