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Okay, why don't we get started? So this is methods in lab research as a topic. Most people find quite boring, but it is quite important to know about. And I'll mention why that is um as we go on. So to start with some learning objectives, we're going to talk about a lot of lab techniques. Essentially, I'm not going to go into too much detail this. Um but we will talk, discuss the different types of uh does that are available and cons of them. So the point is essentially to give you a few of the tools to be able to read and analyze some research papers that you're probably coming across um during your, your studying or your jobs. Um And eventually you'll be able to apply this to your own practice and research in the future. So um like always we're going to start off with a little mental meter quiz. So let me just get that started. Okay. So if you could use that link and go to menti dot com, um we'll get started on the quiz shortly as soon as a couple of people join. So they're, they're, they're a bit tricky questions, but please join because hopefully you'll be able to get something from this and I'll be able to see that you have gotten something from this. Give it a couple of minutes for people to join the quiz. Okay. Give it another minute. It's not too difficult of a quiz. Please don't feel too shy. Okay. Why don't we get started? Just because we don't have too much time. Sure. There's no point for speed. So take your time. There's no rush of 50 50. Not bad. Um Let's keep going. Hopefully this will all be a bit more clear after we go. Okay, let's get started. Perfect. Um Yeah. So I know that might have been a bit uh difficult because a lot of people probably haven't heard of these techniques, but as we go through it, it'll start making a bit more sense. So let me just share the slides again. Okay. So we're going to go through um a few different techniques, but they all have broad themes. So that's what I'm going to talk about in the overview. The different methods can usually the ones we're going to be talking about today may are used to detect DNA or RNA. So nucleic acids to detect proteins or two detective and enzyme is working or not. Um And please, if you have questions during the presentation, put them in the chat and we'll have time to get to them at the end. So we'll start with detecting DNA and RNA and are only one here for. Now is PCR, I'm sure a lot of you have heard about PCR. Now, after, after the pandemic, it was everywhere. So what PCR essentially is, is using, working out whether a particular strand of D N A is present in a sample or not and how much of it. So it uses something called, it uses a DNA sample and something called a polymerase enzyme to amplify whatever DNA you have in the sample depending on what sequence of DNA you're looking for. So if the sequence is present, then the D N A will be amplified, something else called running a fluorescents. The more fluorescent you have the more initial or you can also use the time to reach a certain amount of fluorescence. So if you see in the middle diagram there, you have these sigmoid all type curves and it has something labeled on it called a threshold. And you might have seen this, if you did a PCR test for COVID, it would tell you your CT value. The CT value essentially tells you the number of cycles that was required to reach the threshold value and the more D N A you have at the start. So the more COVID you had, the less time it would reach take to reach the threshold because there's less cycles of amplification needed for the fluorescence to be detected. I hope that made sense if it didn't please do put the question in the chat and I'll re explain it later on. Um So yeah, you can look at pathogenic load using PCR and it's incredibly powerful because you can multiplex the technique quite heavily. So if you have different primers, so primers of the components which are needed to start a PCR reaction to get the preliminaries enzyme to even work in the first place. If you use multiple primers that are, so that corresponds to different sections of D N A, you can get different, you can get, you can check whether all those fragments of DNA are present all at the same time. So imagine you've taken a bacterial sample from a patient and you have a general idea of what sort of bacteria might be present or might be infecting the patient. You can use different primers for the D N A of each of those bacteria and use them all at the same time and see which one pops up and you can differentiate between them using the different size of uh the PCR product essentially. So what's amplified uh And you can actually tell which bacteria is present much faster than regular culturing. Um You can do that for viruses and pretty much any pathogen. So it's quite a powerful technique to use both in lab studies and in clinical medicine. In lab studies, it's mainly used to check whether a gene is being expressed or not or how much of A gene is being expressed. So you can even use this to detect R N A. Um And so, of course, if you have more expression of the R N A, you'll get more of a fluorescent value or a lower CT value. Uh then you can compare that between different samples. So let's say you have a cancer cell which is over expressing a certain gene. Let's say telomerase that one of the telomerase genes. If I do a PCR for the telomerase, RNA A, it'll be much higher compared to a normal cell. So that's how you can differentiate whether a certain cell line or certain samples are expressing a certain gene or not using PCR. So it can be applied in quite a few different ways. It does have its disadvantages though it can't, it can't really be used to detect single nucleotide changes because the primers are not super specific. It can be in some cases, I'm not saying it's impossible, but you would normally use some other method for that. You must know the sequence you're looking for. So like I mentioned before, you have to have a general idea of the pathogen or the bacteria you're looking for because that primer has to be designed to connect to a specific sequence in the D N A of the organism. So you have to know the sequence in order to be able to design the primer to connect to that sequence and it can be too sensitive. So, I'm sure you all heard that after uh 90 days or two months of having a COVID infection, you shouldn't have done a PCR test. That's because any remaining DNA from the remaining dead particles of COVID, those would have resulted in a positive PCR test just because it's so sensitive and even the tiniest amount of remaining DNA or RNA can result in a positive test. Um And some sequences can actually be too complex to, to amplify, but there's a lot of pros. So it's very sensitive, cheap and quick and scalable and multiplex A ble like we were talking about. Um that's why it's becoming more and more common to use PCR, especially in uh labs in the hospital, use PCR for a lot of purposes there. Now, so that's detecting DNA and RNA. I'm sorry, it's going through quite quickly. Uh But we're not going to go through all the details of actually doing a PCR test. So this now we're going to focus on the bulk of our, of the presentation detecting proteins because that's done very commonly in the lab. And in clinics, it's a Western blood. Any, if you have heard of western blood, you'll probably know that people hate western bloods um because they can be very finicky. But what is a Western blood? It's a method essentially use an antibody which is attached to a specific molecule and we know antibodies are super specific. So they'll go and bind onto your protein of interest and that other molecule attached to the end of the antibody will fluoresce or give off some sort of signal that can be detected. So, normally, what you would do is run your protein sample in a gel electrophoresis which separates the proteins by size. And then you use your antibody to detect the protein. So not only can you tell if a protein is present or not, you can tell it's approximate size and you can also tell how much of it is present. Um Obviously, we're not going through all the little details of doing the western blood, but hopefully, this explains its purpose and it's general principles. So you can see this diagram here, they have cyclone be one and they put different masses of cyclone be one in that gel electrophoresis. And you can clearly see the thickness and how dense the band is decreases as you decrease the amount of the protein. And that's you use that as a standard curve to then determine how much protein is in a new sample to the ones. On the right hand side, the labels on top, just refer to different cell lines. So you can use that to extrapolate or interpolate how much protein was in your sample of interest. So it's quite a clever technique like that because it uses antibodies which makes it very specific, but it is very antibody dependent. So your antibody has to be good which leads us to the pros and cons. Um that left hand image at the bottom is quite sad. Um And it happens all the time, the bad and the ugly, I mean, so it's very time consuming labor intensive. It takes about about two days to do an entire Western blood procedure fully. And it's not always very quantitative because you can see you can get all these artifacts in the Western blood itself and a lot of people report these blocks very poorly. So if you look on the right hand image, they've sort of screen shotted out a tiny portion of their Western blood, maybe. And that's what they're trying to move away from doing that. But essentially, the problem with that is you don't know what else was in the rest of the blood. The antibody might have been detecting a whole load of different things there, which they've just decided to omit, which is not great. So it can be reported quite poorly. And we talked about different chances of error and that the antibody should be really good. But we did say it's great in telling you the size of the protein. Um and it's used to verify anti and you can detect antibodies themselves. So the antibody itself correlate to PCR because like we said, PCR is used to determine expression, but that doesn't mean your protein is actually being produced. So use Western blood to determine whether the protein is being produced. And you can check whether it correlates to the over expression of the RNA. So if you get high R N A, hopefully you get high protein and vice versa. So you can look for that. And that correlation is quite nice to see in a paper. The next one up is Eliza and it stands for enzyme linked immunosorbent assay. And it's essentially a very similar principle. It uses antibodies to detect proteins. Um but it's a lot more quantitative. So it doesn't separate things based on size. It's just a one part reaction. And again, it uses fluorescents or colors to detect the change. So you can see in that little plate in the middle, the more of your protein that's present, the more antibodies, what mine. So the more signal you'll get and you can get a nice little curve telling you how much protein was in the sample. And that's actually how COVID lateral flow tests work. They have sort of uh tethered antibodies on the on the chip which then detect your COVID uh particles. So it can do all of this. And it's a lot more quantitative than Western blocks. Simply because you can directly measured the fluorescence. You don't have to use a computer program to do anything. Um There's not a whole lot of steps involved. So there's less places for human error to occur. Western blood has at least 20 steps in it. Whereas this has about six lakh for your steps uh to carry out itself, it does have its negatives because you need to verify the antibody before by western blood because otherwise you have no idea what the antibodies binding to because you can't see it for yourself on the block, for example. So let's say your antibody is not very specific, then you could have other proteins in your sample that bind to the antibody and give off a positive signal, which would lead to a false positive. So you always need to verify the antibody before using Western blood to make sure it's specific for your protein of interest. So it can be a little bit time consuming in that way to verify the antibody beforehand. And you need really, really good controls because your substrate by itself could cause a signal, not because of the binding or anything your antibody by itself might cause a signal. Your antibody could get stuck to the well and create a signal without the protein actually being present. So you need a lot of different controls to make sure what you're detecting is actually your protein of interest. And all of that can take a bit of time. But the best part about Eliza is that it's very quantitative because all you do is read it using a plate reader, we'll get onto plate readers in a little bit. So the last method of detecting proteins is probably the coolest but most difficult to carry out immunofluorescence. And I'm sure a couple of you have heard about immunofluorescence, immuno for antibodies, fluorescents for fluorescence. And it's used to detect proteins in cells. So you can actually localize where protein is. So if you look at the middle to the green panels, they're looking for p 53 which is a tumor suppressor gene in your cell. And what you do essentially is you, you fix the cells, you can't use immunofluorescence on live cells because you need to force antibodies into the cell to bind to your protein of interest and then fluress. So you permeable eyes, the cell get your antibody in which is a bit tricky sometimes. But then your antibody binds to the protein P 53 in this case and fluorescence. And you can look at that fluorescence under a microscope. You can see exactly where protein is. That's quite important sometimes because sometimes proteins can be MS localized. So they might go to the wrong place, which is, which will be the reason that they're not carrying out their normal function and which can eventually cause disease. Um You can actually see their CML up their chance for chronic myeloid leukemia. And you can see that p 53 is what there's more p 53 in the first place and it's in the cytoplasm, those black circles in the cells are the nuclei. Whereas in the normal cell line, there's much less P 53 you can't really tell properly whether it's in the nucleus or in the cytoplasm alone. But yeah, you can, so essentially from this image, you can tell you can also decide on whether there's more of a protein in certain cell line. But it's hard to be very quantitative about it. It's hard to put numbers on them because microscopes very day on day, the weather, all of that uh affects how microscope works. You do have very, very good microscopes which can be directly used quantitatively. But a regular old microscope in a lab won't be too good at doing that. So again, that's a disadvantage. Heart um results can vary hugely having done this myself, you get very different values on different days for immunofluorescence and using different software to detect the fluorescence. So it's not the best at being quantitative and it tells you nothing about the protein. You don't know how big the protein is. Um all you know is where it's hiding. Um And if it's very low expression microscopes aren't very good at dealing with very low levels of fluorescence. So sometimes you might do immunofluorescence and it'll be a blank screen. You're gonna think, oh my protein isn't there. When in fact, if you do a western blot with the same cells, you will detect your protein because the way the antibodies in a western blood work is that they amplify the signal. So even if you have very few molecules like you saw in that image, there were nanograms of protein. Even if you have very small amounts, the signal gets amplified enough to be detected by the system and your antibody must be good. You don't know if it's actually binding to your protein of interest. So again, you need to verify it beforehand using Western block. So you can see Western block the whole theme here. Um but it does have its pros, you can see where the cell proteins are and you can sometimes quantify changes. So all these methods were to detect the different kinds of proteins um that you might want to look for. Why might you want to look for different proteins? Well, you can have diff over expression or under expression in different conditions or cancers or maybe you've, you've introduced a new protein into a cell line and you want to see if it's being expressed, then you'd use one of these methods. You want to see where it's being expressed. You do you use immunofluorescence? So there's quite a few different reasons. You need to look for proteins themselves, you know, proteins are the building blocks of life. So you do need it. Um There's one last method called flow cytometry which some of you might be familiar with. It's a bit more common in clinical medicine because it's used. Well, let's talk about what it is first, it's a single cell method. So it looks at each individual cell and depending on what you're looking for, you, you can use antibodies to bind on two different molecules on the surface of the cell or inside the south. If you're looking on the surface of the cell, you can actually keep the cell alive because all you have to do is add the antibody to it. It's not as simple as that. But in general terms. So you can actually use live self. So flow cytometry, which was one of the questions in the previous quiz, you can use lifestyles and you can look very, very quantitatively using detectors in the flow cytometry machine to see how much of a protein is present on the surface or inside. So in this diagram, you can see each little dot It's a bit pixelated but each little dot represents an individual cell in your sample. The X axis shows the expression of a surface marker called CD 34. Um And the y axis shows the expression of CD 117. I'm not entirely sure if they've used antibodies here or what they most likely have used antibodies which will bind onto those markers and fluoresce which can then be detected by the system and it will place your cell in one of these four quadrants that have been separated. So if you're so yeah, essentially tells you the amount of fluorescence of those cells and it's extremely powerful because it's single cell. So you can get a much better idea of your sample rather than the other methods. Besides immunofluorescence. Actually but it's much more high throughput than immunofluorescence. Because even though immunofluorescence looks at single cells, you have to manually move the slide around to look at each cell. Whereas here, the machine just does it all for you. Whereas Western blot and Eliza or what we call a bulk method. So it's looking at all the cells in your sample were analyzed at the same time, you can't distinguish how much protein was in each individual cell. But flow cytometry can do that and that's very cool. Um So you can look at protein expression Purcell and you can count the number of cells. It allows you to do a few other things as well. Like look at the shape and size of a cell, the internal complexity and granularity of a cell. And what it's commonly used for is to analyze, well, in, in the clinic, it's used to analyze blood samples for things like leukemia. You can look for specific surface markets on a cell and because there's certain surface markets are indicative of certain types of leukemias or lymphomas and you can look for them using flow cytometry. Another useful purpose of them is they can actually be used to separate cells. So if you have one population of cells which express CD 34 1 population, which explores C D 117 and they're all mixed up together, you can use the flow cytometer and tell it that if my cell is expressing CD 117 and not CD 34 put it into this vial if it's expressing CD 34 but not C D 117, put it into the other vial and the machine will literally separate the cells for you to get two completely different populations. So it's a very, very commonly used tool nowadays and the data looks exactly like I've shown you up front. It's, you'll see this sort of scatter plot and the way you interpreted is using the axes to see how much fluorescence was coming corresponding to that specific uh antigen. Okay. And the pros and cons, I think we've already talked about the pros quite a bit cons, I've already talked about how hard it is to get antibodies into cells. And the other thing is cells can auto fluoresce which can sometimes confuse the system, but it has things that it can do to deal with that cold competence. Well, you have to use the correct controls to normalize for the auto fluorescence. So cells which haven't been targeted bit the antibody because then you can measure how much they fluoresce just by themselves and cancel that out from your actual samples. But you can also do something called compensation. Close cytometry can also be multiplex heavily. You can look at like 50 hundreds of different antigens if you want to. But because it measures fluorescence, the fluorescent spectra for different antibodies or different uh markers can overlap. So the system needs to be able to sort of separate them away and say that this signal is coming from this antibody, not because another antibody is sort of leaking into the signal range and that's called compensation. And if you, it's quite hard to do manually, so good flow cytometer is do it automatically, but it takes a bit of time and fiddling to get it right. But if there's any mistake in the compensation, your results can't be interpreted. So you have to have a really good technician to help you do your experiment to make it look worthwhile. So if they haven't mentioned compensation in their paper, don't trust it. Finally, we're going to detecting enzyme activity using a plate reader and most enzyme activity essays just um have a very similar principle. You add your buffer in which the enzyme works. You add a substrate which will fluoresce or uh change its absorption absorption or emission spectrum when it's cleaved or when it the enzyme works and you add your enzyme. So enzyme cleaves substrate substrate releases a signal signal is detected by something called a plate reader which can look at fluorescence, you can look at the spectra um and quantified. So, again, very quantitative. Um and you can, this is what's always used to look at the enzyme activity. There's a couple of other methods, but we're not going to go into them right now. And the PROzac is quick and easy. Like I mentioned you can do 56 of these in a day. It's very easy. But, but it's always about, you have to have very good controls. Something like you have to look at the substrate alone with the buffer itself just to make sure that the substrate isn't being cleaved just by itself without the enzyme present because that would lead to a false positive result. Other ones which are not as important or you want to look at the enzyme by itself in the buffer just to make sure that it's not giving off some sort of signal that's being detected by the plate reader, which might interfere with your experiment. And you need to know the nature of the substrate. Well, you want to make sure people have used it before to see if it's reliable or not and different substrate will admit different types of light. So you need to make sure you have the correct light filters or wavelength filters for your plate reader and they can be quite expensive. So you should check that before buying the substrate for the experiment, which I made the mistake of not doing. But luckily we had the filters. Um So in summary, that was a lot we've gone through and I'm sorry, it was if it seemed a little bit quick, but you want to how to approach a lab problem or a lab question or even reading a paper is think about what you want to show. And what is the least limited way or the simplest way of doing that? And when you're reading a paper, you wanna, before even reading their methods, you want to read the title of the paper and think about okay, if I had to do this experiment, what method would I use? What's the most reliable and what would I trust the most? And then you read the paper and you have a look at what they did and you want to make sure you know why they've used a specific technique because all techniques have their limitations, like we said, does their experiment really show what they're, what they're claiming? It shows and sometimes they'll draw conclusions from experiments even though that's not a conclusion you can draw from the experiment and how you look for those is knowing the pros and cons off the technique and how the technique works in general. So now you should have the skills to read through a paper and be like, okay if they've used the western blot here, can I really trust their quantification of the Western blood or they've don flow cytometry like we're talking about? But they haven't really mentioned compensation. Can I trust their flow cytometry results? And that's how you need to be thinking when you read a paper. It's basically trying to, well in the cruise is trying to savage their paper and try to try to come up with reasons why the experiments might be flawed or their results might not be true. Um And you also wouldn't want to look for cherry picking of data like I should in the western blood. You don't want to really trust. Well, a lot of older papers will have it and you have no choice but they just had that slim band showing only their protein of interest. You don't even know if that was at the right sort of size for their protein because you don't have the entire blot. Do you want to look for cherry pick data and be quite cautious about interpreting it? And then you can also go step further, you can see how you can improve medical practice by applying some of these techniques in different areas and maybe do a project on it. A lot of people ask in the um form beforehand that they don't have the knowledge or they don't know how to start on a project, propose a project like this uh to sort of broaden the use of PCR in a lab in clinics and then see whether that actually makes a difference. So there's a huge amount of potential here with the things you can now read a paper better. If you want to do a lab project, you're a bit more educated on what techniques you might want to use or learn about further and you can sort of propose these projects to the supervisors. So yeah, that's only the tip of the iceberg. I've gone through the most simple techniques and not even the actual process of doing them so well. If you guys want, we can do a future session on specific methods um going into a bit more detail, but we can, that'll come in the future. So the last bit, I want you to post any questions you have in the chat. But what we're gonna do now is the final mentee quiz and then we can come back to the questions you posted in chat. So I'm just gonna share screen of the post uh webinar mentee quiz and we can quickly get started on that. Just share my scream. All righty. So you have the code up there, please do join the mental meter quiz. Hopefully, it's a lot more. Well, hopefully it's kinder now since you have a general idea of what's going on with these different techniques. So I'll give it a couple of minutes. We're actually doing pretty well for time, which might mean I've rushed through slightly. But if that's the case, this presentation will be available later on for you to have a look again. So I'll wait a couple minutes. Okay, perfect. We have three people in the pre quiz. So I'll just get started. Now um again, there's no point for speed. So just have a go. So we're looking for something in common between all those techniques. Look at that 100%. My something has come across. I'm glad. So next question. Oh, excellent. We have another player. Join us start. It's a bit trickier. But how'd it go? We did talk about this specifically actually. So hopefully you guys remember. Yeah, majority wins. So you wouldn't use the enzyme alone with water because water is probably not what you've done the experiment in because enzymes need specific conditions to work and those conditions are provided by a buffer. Um But yeah, you would use the substrate alone with the buffer because like we said, if the substrate is breaking just by itself in the buffer, without the enzyme, then you're going to get a false signal being produced. And last question of the day. So by CLL, I mean chronic lymphocytic leukemia and CD 38 is a surface molecule which is present on cells in chronic lymphoid leukemia. Oh 50 50. So you would use flow cytometry just because it's a cell surface molecule. And what we said is it's much quicker uh at looking at self surface molecules. And it's most importantly CD 38 is a cell surface protein. And PCR is used to look for nuclear it, acids of D N A or R N A theoretically, you could look for expression of the CD 38 gene in see in chronic lymphocytic leukemia. But it makes it a lot more complicated and it would take longer because you need to have a sample without CLL to verify. Whereas this just looks at expression, you can have a threshold. Okay, perfect. Good work guys. It looks like um you've learned or taken away something from this webinar. So let me just share the slides one last time for any questions. Um While you type your questions into the chat, I'll just put up the QR code for the feedback form and I'll be monitoring the chat. So if any of you have attended the previous sessions, you know, I normally put also put a link for the next session, but we haven't organized that yet. So that will be up on Facebook as soon as possible. So nothing coming in on the chat yet. I will also put the feedback form in the chat and please do fill it in because that's how you get your certificate at the end. So I've also put the link to the feedback form in the chat so you can access it either way. So see there's a few more people now, I didn't notice them on the chat before, but hopefully you didn't miss too much. Uh But if you do have any questions, please place them in the chat, I'll be here for a few more minutes. So take your time if you got anything wrong or I rushed through something, just let me know we'll go through it. It looks like I did such a good job. No questions or it was just so complicated, but no, you guys answer the questions really well. Yeah, I I do understand. If you don't want to ask any questions, it can be quite tricky to ask questions on a very technical topic like this, especially if you're learning it for the first time. So if you do run into any questions, do put it in the feedback form uh later on, or you can message mind the bleep on their Facebook messenger and I can clarify your queries there. So if there's no more questions, uh if there's no questions, feedback link, the feedback link is in the chat, uh I can repost it in that if you'd like. Uh I just re sent the feedback link there. So hopefully that works for you now. Excellent. Um If that is all, we will finish off for today, please do do the feedback eventually and I'll see you at the next session. Hopefully um should be a bit more clinical on that one. So if any of you are interested, it will the add will be up on Facebook soon. Bye guys. Thank you.