Discover Real-Time PCR for the Classroom

hello and thank you all for joining us today for our overview and introduction to using real-time PCR in the classroom I’m Ingrid Miller product manager for bio-rad’s biotechnology Explorer program I’d like to introduce David Palmer our real-time PCR expert and presenter today a study at that excuse me Dave studied at the University of Delft and Ontario Canada where he received his Bachelor of Science in agriculture after receiving his doctorate in molecular plant pathology from Oklahoma State University they’ve performed field and greenhouse research on biotech crops for Zenica AG products he then co-founded a biotech startup company called biotic currently Dave is a senior technical support consultant for bio-rad laboratories supporting educational electrophoresis gene transfer and PCR products they’ve is very passionate about education and teaches an annual PCR course as an adjective at Contra Costa Community College come Dave alright and thanks everyone for attending this real-time PCR presentation that we’ve got today is the audio level okay if the person participants can click on the yes check box if you can hear me okay all right good good okay so we’ll continue on them just clear those check boxes so today what we’ll talk about is real-time PCR and a couple of different things first we’ll go over what is real-time PCR used for it how does real-time PCR work but chemicals and instruments are used to detect DNA but does real-time PCR data look like I couldn’t lead demonstrate real-time PCR in the classroom and what are some ways to troubleshoot wheel bill time PCEHR there just a little bit of housekeeping to start off with if you have questions about prep their presentation we’ll try and answer them at the end of this so questions you can send by text chat to the presenters and at the top of your screen you’ll see a little text chat box and you can just open that up anytime you’d like and sends them send some questions and if there’s anything you want to know about whatever we don’t get answered live at the end of this presentation I’ll try and answer by email and that will be attached to a a mail that will be sent out after after the president so to start off with what is real-time pcr and what is it used for well many people here have taught PCR before and we remember that PCR is the polymerase chain reaction it’s a process for the amplification of specific fragments of DNA so amplification and specific are the two key components they’re real-time PCR is a specialized technique it’s part of regular PCR but allows a PCR reaction to be visualized in real time as the reaction progresses so as we’ll see real-time PCR allows us to measure minut amounts of DNA sequences in a sample and that the basic summary of this whole presentation is right here conventional PCR tells us what real-time PCR tells us how much so some examples of things that real-time PCR is used for our gene expression analysis for cancer and drugs your research for example disease diagnosis and management food testing used an animal and plant breeding and also forensics and I’ll go into a bit more detail on some of these one example for the use of real-time PCR and gene expression analysis or basic research would be research into the brca1 gene that’s the gene that’s involved in breast cancer and that gene is involved in tumor suppression it modulates the expression of other genes so way the brca1 protein works as it affects the conversion or the transcription of DNA to messenger RNA and that ultimately affects the amount of particular gene product protein that’s produced real-time PCR will tell us how much of that messenger RNA is produced from a particular gene so when researchers do gene expression studies and say that a certain treatment or a certain disease state up regulates or down regulates a gene a lot of time they’re getting that data from real-time PCR because it tells

them how much DNA or messenger RNA there was another use for real-time PCR is in disease management and an example for that would be a typical HIV treatment and in in that example what the physician would do would be to monitor the viral load or the amount of virus in a patient and real-time PCR allows us to do allows them to do that by looking directly for the amount of virus RNA in the blood the real-time PCR will amplify the RNA and quantify it and you’ll be able to adjust prescriptions and disease treatment based on the amount of virus as determined by real-time PCR and another example kind of related to my background with work with with the zenica is in determining the percentage of GMO food with genetically modified foods those foods that have a foreign gene inserted into the a lot of countries have regulations as to what the maximum amount of that type of food allowed is and the way that you can determine how much GMO content for transgenic content is in a certain shipment of grain for example is to sample that shipment measure the amount of regular or wild-type DNA I have in this slide WT DNA for their regular wild-type DNA also measure the amount of genetically modified or foreign DNA and if you take that ratio you can determine the percentage of GMO food and that’s the common leeway used to measure how much of a shipment contains genetically modified food or not you know yet another example of real-time PCR that’s just kind of coming into play is real-time PCR and forensic analysis there are some real-time techniques that can be used to identify stains so for example a stain on a carpet rather than using a color change test like you’d see on the TV shows where they put a q-tip on the stain and then add a reagent and see what color changed to determine what that stain was made of real time PCR can actually help determine what what the composition of that stain is whether it’s human blood or animal blood for example another factor that relates to friends at Canal ELISA would be how much DNA is in a particular sample because when the DNA is subjected to genotyping the genetic fingerprinting a certain minimum amount of DNA is needed and real-time PCR can help to determine whether there’s enough there and how much is there so those are some examples of a real-time PCR now we’ll go on to how does real-time PCR work and in order to understand real-time PCR we’ll just review how regular regular PCR works and quite a few of you have already taught PCR in your classes regular PCR you start with the double-stranded DNA template you also have in your PCR reaction or your PCR tubes the primers and nucleotides and the DNA polymerase along with buffer and water and all the components of the PCR reaction that reaction tube is then heated and a thermal cycler and when it’s heated the DNA denatures once that reaction is cooled so the primers can anneal the primers will bind to their specific recognition sites and then the TAC polymerase that’s also in that reaction will go and extend those primers with the nucleotides and make copies of the original templates so when we start with one original template by the first reaction we end up with two after who repeats that cycle we’ll have four copies of our original template and then after three cycles we’ll have eight copies of her original template so PCR goes on in the next financial manner and you know many of you have taught PCR already and you’re familiar with that when I teach PCR at the local community college and teach over at Contra Costa Community College I often use a nice little animation that bio-rad has it’s got a lot of detail on it it’s very well drawn and very very clear and also scientifically accurate which is kind of nice so it’s my favorite animation there’s also animations available on the internet like one from Cold Spring Harbor I also use that has some good information on it and there’s also a new PCR poster that fire at has come up with in one poster has from beginning to end how PCR works and I’ll be using that next spring when it when they teach anything that’s a really good resource

to have so that’s how traditional PCR is usually presented and in order to understand how real-time PCR works what we’ll do today is use a thought experiment and this is kind of a different way to teach real-time PCR than the way I learned it I had seen presentations before on real-time PCR and they’re really hard to follow and they didn’t really make sense for or how you would relate that the information coming off the computer screen to quantity so what I started doing after I had bad luck teaching that to my own class was I switched to this method and it seems to work really well so think about this if you’re teaching real-time PCR for for your own class you may want to try this method so this this is what we do to understand real-time PCR what we’ll do is we’ll imagine ourselves in a PCR reaction to but cycle number 25 so we’re there in the PCR – what’s in that PCR tube well in that tube what we’ve got our nucleotides we’ve got primers we’ve got our original template that we’re trying to detect we have amplicons the amplified DNA product we’ve got our enzyme and we’ve got the buffer and the water and other things in there and just for the sake of argument then let’s say we’ve got 1 million copies of the amplicon right now cycle 25 and we’ve amplified and amplified and we’re now up to a million copies so continuing in this thought experiment what was it like in the last cycle at cycle 24 oh the conditions for almost the same as what we are now at 25 but we only had half a million copies of the amplicon make sense right because PCR doubles every reaction though if we go back to the last cycle we only had half as much what about cycle 23 well cycle 23 we probably only had 250,000 copies and then cycle 22 cycle 22 is likely the same reaction in the tube but only a hundred and twenty-five thousand copies of the amplicon so what we can do is we can plot that on a graph and if we plot the cycle number on the x-axis versus the quantity of DNA on the on the y-axis at our first point right there cycle 25 we had 1 million copies cycle 24 half a million twenty-three hundred twenty-five thousand and so on we get this nice exponential curve and that’s exactly what you’d expect from PCR we’ve got that working backwards but also work to it forwards where you’re amplifying an amplifying you can’t see it on the graph because of the scale on the graph and then all of a sudden you you see the curve shooting out so now continuing this thought experiment that’s going to happen after the next cycle cycle 26 well cycle 25 we had 1 million copies cycle 26 or probably of 2 million and what about cycle 27 well if we had 1 million at 25 2 million we’ll have third cycle 26 off 2 million cycle 27 we may have 4 million copies we can probably see where I’m going with us what’s gonna happen at cycle 2 Hackel 200 in theory if we kept doubling there’d be one with that many zeros I think that’s around 69 zeroes numbers of amplicons that works out to 10 to the power of 35 tons of DNA and that much DNA would be the equivalent in weight to 10 billion planets the size of Earth so that’s a huge amount of DNA and obviously that’s not going to happen so have this note here a clump of DNA the size of 10 billion planets won’t quite fit in there PCR tube anymore there’s no way and realistically is the chain reaction as PCR progresses it gets harder to find the primers it’s harder to find the nucleotides and the polymerase enzyme itself is wearing out too so there’s all sorts of limiting factors that will come into place keypoint exponential growth does not go on forever which is good so if we plot that part of the curve we have continued exponential growth and then we hit a plateau as reagents run out and this basic curve shape sigmoid curve shape is common to every real-time PCR reaction and it’s common to every PCR reaction period PCR is a doubling the doubling will keep going and then it will stop and that makes a sigmoid shape when you draw it so once we’ve seen that we can relate that to quantities and here’s how we will think about that using the same thought experiment what we’re gonna do is we’re gonna imagine that you’ve got a

tube of PCR reaction similar to mine but your tube the green tube you started with four times as much DNA as I had in it and we’re gonna picture our tubes both in cycle 25 and work backwards so my tube is the red one cycle 25 it’s got 1 million copies your tube is degree 1 at cycle 25 let’s say it’s got 4 million copies we started with 4 times as much at cycle 25 where we are now you you’ve still got 4 times as much if we look backwards so cycle 24 you had 2 million cycle 23 you had 1 million and note there that at cycle 23 you had the same amount of DNA that I’ve got at cycle 25 the only difference is it took me 2 cycles to make up for that difference do doublings and i made up for your four-fold lead on that a way to look at this is with our graphs and the original graph I showed was the one of my read PCR reaction Chambal fied to 1 million copies at cycle 25 you started with 4 times as much so for every point on the curve you’ve got four times as much what that means is that you’ve got amplification essentially two cycles earlier your curve is the same shape as mine but it comes up two cycles earlier because you’ve got a head start of two doublings we can look at this another way to that if I’ve got my redtube cycle 25 with 1 million copies if you’ve now got a tube of PCR reaction it’s green dye in it and it has eight times less DNA and then at cycle 25 it’s only going to have one a as much but then cycle 26 it will double 27 it will double 28 it will double again cycle 28 we’ve got 1 million copies you’ve got the same amount as I had back at cycle 25 so it took you 3 cycles but you caught up to the same 1 million copy number I did and we can plot those curves on a graph as well my original red PCR reaction amplified is that the red curve year to the blue PCR reaction had only 1/8 the amount of DNA after 3 cycles that had caught up to my amount of DNA so the red and the blue curves they’re identical in shape they’re both the sigmoid shapes the only difference is they’re shifted left and right from each other yours had less DNA in it so it took a little while longer to catch up any individual point on that curve so what we can see from this is that it’s the left/right shift of those curves is related to the starting quantity of DNA more DNA you start with the earlier those curves are going to appear and the less DNA you start with the the later they’ll appear because it takes more doublings whether those curving Irv’s to cross any particular point and we can quantify this particular position of the curve by what we call the the CT value or the cycle threshold value and that’s where the curve crosses any individual arbitrary threshold so in this example here our threshold is a 1 million copies in chameleon copies of DNA it’s where our PCR reaction finally passes the 1 million copy mark and one of our reactions it passed at cycle 23 another one passed at cycle 25 another one took 28 cycles to reach that point with those CT values those can tell us the quantity right there is the quantity it’s related to 2 to the power of the CT value every reaction cycle every cycle is going to be a doubling of the cycle number just tells us the quantity based on dublin’s and like a lot of things in math or biology we can plot these on a graph and if we plot or read PCR rowanda T of 1 and a CT value of 25 on the y axis you can plot our green PCR tube that had a quantity of 4 this is a log scale on the x axis right now and a CT value of 23 and the blue tube had a quantity of about 1/8 and the CT value of 28 see that all these points these 3 stars fall in a straight straight line and that’s true with real-time PCR reactions you do a standard curve in this format and all your standard points are generally going

to be on a straight line for any unknown sample that you have for example if we had an unknown that amplify it and cross the threshold at cycle 26 and we could read right off the x-axis what the quantity is and that’s how real-time PCR is used to measure quantities just a standard curve and then compare the CTS of your unknowns to your standards now one thing I like to share with my classes is how sensitive real-time PCR is and keep in mind that real-time PCR is just as sensitive as regular PCR so ultimately even a single copy of DNA can be measured but in reality it takes about a hundred copies to get a reliable signal so you know you’re not just amplifying primer dimers or contaminants or something like that and an example of what a hundred copies would be is twenty atta grams of DNA and not often we use the prefix a dope but that’s what it is twenty atta grams or if you were to work that out and figure out how much blood that would be in a blood stain that would be two one-hundredths of a microliter of blood would contain about a hundred copies of any particular gene so we all know how small a microliter is picture just one fiftieth of that and that’s still easily detectable by PCR and real-time PCR so it’s kind of cool to share that with students see if they like to see that part so that’s kind of the theory of real-time PCR now how do we actually find out how much DNA is in that tube as its amplifying what we use our reagents that fluoresced and the presence of amplified DNA so we use fluorescent reagents that will only fluoresce when they’re near their PCR product and we’re all familiar with one of those products or one of those one of those reagents idiom bromide it’s common and it’s well known but we also know it’s toxic and it doesn’t happen to be very bright in terms of fluorescence so xxx and bromide what it does is it intercalates into DNA so it gets in kind of flat ways between the stacks of the DNA bases when it’s in that position that does is it becomes fluorescent so you can shine a UV light on your gel and your bands will wear s because the sitting bromide is bound in there and it’s a fluorescent like that that also makes it mutagenic due because it doesn’t come out of the DNA that easily and it will really interfere with the enzymes that are trying to act to copy the DNA at that point now an alternative reagent that we could use in real time PCR and this happens to be one that’s almost always used compared to idiom bromide anyway is called cyber green one it’s used because it’s got very bright fluorescence it doesn’t think much dye and it gives a very very bright signal way cyber green works is binds to the minor groove of the double-stranded DNA so rather than getting inside flat between the basis it sticks on the outside of the double strands of the DNA and because of that that actually makes it much less mutagenic as well it’s hard to see the details in this little graph but the red bars are the mutagenicity of a vidiian bromide tiny little green bars on the bottom that’s the mutagenicity of cyber green so cyber green is still mutagenic because it interferes with DNA but much less so than if idiom bromide the main reason it’s used in real-time PCR though is because it’s got great fluorescence so as real-time PCR makes more double-stranded DNA the cyber green fluorescence more here’s a slide that kind of shows that as the PCR reaction goes along and you go from double-stranded to single-stranded DNA and then more double-stranded copies to have cyber green but binds to the double-stranded DNA that cyber green is illuminated by light of one wavelength will fluoresce in another fated flank so for example if you hit it with UV light it will fluoresce a bright green color and real-time PCR machines can can do that they don’t use UV light they use visible blue colored light but they’ll still measure the green fluorescence so how do the instruments do this well real-time instruments consist of three major components one is a thermal cycler PCR machine that does the actual amplification of the DNA by raising lower than luring the temperatures another component is the optical module and it will detect the fluorescence in the reaction and then finally there’s a computer and that computer will collect the data coming from the instrument and it will also program the instrument with what temperature’s it’s supposed to reach one example of the real-time PCR machine is our higher ed mini off con real-time PCR machine and you can see

the two components here the upper component is the optical power and it contains an array of LEDs that can loom innate different wells in a PCR plate and they’ll illuminate the reagents in those wells if there’s double-stranded DNA and cyber green and that in that well it will fluoresce and there’s a detector that will detect the fluorescence so it can measure the fluorescence and O or d8 whilst the lower part of the PCR machine is the actual thermal cycler and it just raises and lowers the temperatures the reactions of it it’s kind of the workhorse that carries out the actual thermal cycling of the PCR these two components are together in one machine another example is the CFX 96 real-time PCR machine and it follows the same basic principle you’ve got on the bottom the thermal cycler does the temperature changes and gives you the PCR reactions and then the upper part contains the optical module and in this case the CFX 96 it uses multiple LEDs that can scan over every well of a 96-well plate and DNA fluorescence is then detected with a set of different photo diodes so it can measure multiple different colors at once so researchers can do multiplex real-time PCR where they’ve got multiple colors in the same tube so I mentioned the computer the computer that’s connected to a real-time pcr system we’ll be doing a couple of things the first one is that lets you set up the pcr protocol so you can program in what the temperatures are that you want to each step for the PCR cycle to hit 95 degrees 60 degrees 72 degrees whatever it happens to be the the next part of the software lets you set up the plate and plate set up will tell the software where the unknowns are on the plate where the standards are you’re doing a standard curve wear note no template controls are so it helps us offer analyze the data after and then the software will also show you what the data looks like as it comes in and then after it’s finally collected so typical data that would come out would be actual traces showing the real-time PCR amplification if you have standards you get a standard curve and have a picture of your plate layout and then a table of table of numbers it would represent the CT values that left-right position of the curves so just a reminder if you have any questions about the presentation there are things that we’ve covered so far things that we will cover before the end just send a text chat to the presenters and we’ll try to answer those so now I’m – what does real-time PCR data look like and what are melt curves so this is some actual real-time PCR data you can see it it looks very similar to that theoretical data that I showed you earlier each traces got a nice sigmoid shape there’s amplification and there’s a left/right shift on these curves they’re further towards the left curve is say for example curves here they have a lot of DNA curves over towards the right have a lot less DNA and the reason for that is these curves essentially have a head start they started with more DNA so they’re going to amplify earlier final product of real-time PCR data is essential a table of numbers that the graphs are nice to look at but it’s actually the numbers that let us do the quantitation or the calculations so for example for every well that you have in this case I highlighted well a 3 it’s going to have the the flora for cyber in it it may be a standard and across to the threshold at 8.9 at a CT of 8.9 another well could be this one b6 contain cyber as well as a standard and across the threshold at 18 point 6 9 so without knowing anything else we could do some quick back to the envelope calculations here we take 18 point 6 9 which is about eighteen point seven subtract eight point nine the CT value of the other well that comes up with roughly say ballpark n CT units between the different between the two wells a doubling every CT unit so two to the power of 10 that’s equal to 1024 1024 so without knowing anything else about these wells right away with those CT values alone we can say there’s about a thousand fold difference in concentration between the starting DNA

of this one on the top and this one near the bottom that’s really the core of the calculations that come in real-time PCR is just relating those CT values and the difference between them with a doubling every cycle is calculating it out so that’s kind of the quantity side of real-time PCR another thing real-time PCR can tell us is what it’s not as good as an agarose gel in many cases for telling us what’s in the reaction but it can give us some ideas as to what’s in the tube and that’s the concept of melt curves so melt curves are based on the principle that is a heat a double-stranded DNA it becomes single-stranded so if you’ve got DNA binding dice attached to your double-stranded DNA as it’s heated those binding dyes will fall off and will no longer be fluorescent so if we picture our PCR reaction and after it’s done you’ve got a load of double-stranded DNA it’s cold the DNA is brightly fluorescent because the cyber green for example is bound all over him if we start heating that PCR reaction up now and taking that DNA product and denaturing it we’re gonna have less cyber green able to bind because a lot of the DNA is becoming double striker becoming single-stranded it’s becoming denatured and all ultimately if we will all be single-stranded and no cyber green will bind at all so as we heat a PCR reaction that has cyber green in it what’s going to happen is the fluorescence will decrease as we get the reaction hotter so on this graph here but temperature on the x-axis and the amount of fluorescence on the y axis as we increase the amount of temperature in the reaction the DNA will denature the cyber green will fall off and we get a decrease in fluorescence now what’s neat about this is if the DNA is a different structure in different tubes for example you’ve got a longer PCR product and one tube within another or maybe a different GC content and that DNA that’s longer or has a higher GC will also have a higher melting temperature so it’s gonna take a lot higher temperature before it finally melts and by peaking taking these melt curves from from different tubes what we can do is see if it’s the same DNA that’s being produced in different tubes commonly these melt curves will be presented as the derivative of this so instead of the graph on the left as I showed a ridge linking if you plot the slope versus tip you’ll end up with a trace that looks like chromatograms and that that’s a lot more easy to interpret and I kind of like to bring up mill curves when I teach be scared cuz i got students thinking about the structure of DNA the GC versus a t base pairing how GC takes a lot more energy to separate than an 80 also how a longer DNA frag it’s going to take more energy to pull apart than a shorter DNA fragment so just teaching the concept of milk curse can can lead to a whole lot of different discussions that you can bring out about DNA kinetics and things melt curves right now or have been taken even further with this new field of precision melt analysis or sometimes called high-resolution melt in that a regular melt curve is done with the precision instrument and reagents that bind to every every available spot on the DNA the the melt curve is done it’s then normalized so the starting fluorescence and the ending fluorescence is the same for the melt curve and then it can be normalized for temperature again in the software what ultimately happens with this is for different DNA sequences you’ll get a totally different melt curve totally different shaped curve for different sequences and this can be so specific that it can tell the difference between an A and a T base pair at any given location so it’s commonly used for mutation analysis or mutation screening also it’s used in ecology and and things like that or you’ve got a population of organisms and you want to see how many different species you’ve got in that population say a population of bacteria how many different species are there you can see right away by how many different curves you have so that’s higher resolution Milt so now on to something I enjoy how do we demonstrate real-time PCR in the classroom I usually have the real-time lab is the kind of the last section of my short PCR course that I teach one way to teach real-time PCR is with our

crime-scene investigator PCR basics kit and people have run that kit before what they’ll they’ll know what what you do is you do PCR on some supplied DNA samples we’ve got different suspect samples in a crime scene sample you can run the PCR products out on the gel and see the band patterns and match up the suspect but the I’m seen sample so for real-time PCR you can take that same kit but instead of using the super mix in the kit you can use a cyber green super mix when the contains cyber green in addition to all the other pack polymerase and nucleotides and things you can use that cyber green super mix and a real-time PCR instrument and computer to run the kit so you’re basically just substituting those components you can still take the PCR product out at the end and see the band pattern on the agarose gel but as the PCR reactions are going along you’ll see the amplification happening and that that’s really cool for your students to see the amplification as it as it’s going on we’ve got a application note and a starter kit that are available to help people out with this and basically it’s just running real time as an add-on to the regular regular kit another thing that that you can do with the crime scene kit is to do a dilution series with the DNA since it’s no one DNA template that comes in the kit you can just do a dilution series and see how well the students pipetting is and see what the amplification looks like where their curves come out or for a dilution series because the PCR products in that kit are different sizes you’ll also have different Peaks on your melt curve so you can use that introduce melt curve and DNA structure and things like that so this kit is actually great for first-time users because you get DNA supplied with the kit the primers come with kits it’s all pretty much known to work right off the bat now more advanced users can do the GMO investigator kit in real time and that GMO investigator kit is one in which you’ll do PCR on actual food samples from the grocery store to see whether they’re genetically modified or not there’s primers in the kit for the plant DNA and primers for the genetic law and with real-time PCR you can substitute cyber green super mix for the regular super mix friend the reactions on a real-time instrument and then in addition the agarose gel that you’d normally run to see kind of a yes/no genetically-modified answer you’d also see the amplification curves and those positions of the curves will tell you how much DNA was there ERV comes up early it tells you there is a lot of DNA recovered from one’s food sample if it comes up later it tells you there is much less DNA in that example we’ve also got a starter kit available for for this and an application note and this is one I run in my class and I really like doing this one the ultimate result of this kit can potentially be a table that you come up with or different food samples and what you’ll get is the amount of genetically modified food in each food sample because a real-time PCR goes back to the quantity of DNA you can calculate all the ratios of different things and get those answers so this is a good kit for more advanced users I wouldn’t recommend it as a first time because of the big variability in the food samples that you’ve got so just kind of reiterate teaching real-time PCR in the classroom is it it actually is really easy you can use the crime-scene kit with the starter package or the GMO investigator starter kit for real-time if you’re doing your own PCR say you’ve been teaching PCR using your own primers you can just substitute a cyber green super mix for your ear tack glimmer ace and run it on a real-time instrument there’s really nothing fancy about the real-time other than you’re using a slightly different polymerase you’re also using plastics that the real-time instrument can see through so you can’t use opaque caps and things like that and you’re you’re running there the samples on a real-time instrument so it’s it’s really easy to do you can even design your own primers if you like to get started with there’s really no difference between conventional PCR primers in real time it’s only if you’re trying to get super optimized reactions that need to worry about that so I think real-time PCR in the classroom is cool for quite a few reasons but here are some of them one is instant results you actually see the application within minutes of starting

the PCR run you don’t have to wait until the next lab period for seeing the results and anyone who’s talked real or taught conventional PCR knows that you take your samples you put them into the PCR machine and then the class ends because you don’t have two hours to wait it can continue on so it’s the next day or the next week that you pick up on that with a real-time PCR the students get to see the amplification as it’s going on another thing that is I call the what will happen next factor that’s because they’re the samples are all amplifying at different times you get students who a lot of weight even after class for their own particular sample to amplify so they can see what happens and that really surprised me the first couple of times I saw it were students that would normally be the first out the door they’re actually waiting 15 20 minutes half an hour after class just so they can see their sample curve come come up and amplify it also does what I call demystifying the black box instead of putting the PCR tubes and the PCR machine and closing the door and that’s it now students get to see what’s going on there they see fluorescence increasing in those wells based on what the computer is telling them and even the temperature control of the PCR machine the controlling software will show you what temperature the lids at what temperature the blocks at and I’ve seen students get really interested in just that aspect alone and another thing I’ve noticed is it really does connect computers and biology so students who are into computers I’ve had a number of like Adult Continuing ed students who came from a computer background they get really excited to see the computer control at the instrument and how the computer collects the data and analyzes the data so that it’s kind of my favorite thing to teach just because it does engage students so much and it has those kind of world applicable applicable values students putting on a resume that they learned PCR that’s one thing if they learned real-time PCR that that says a lot more so that’s just some reasons why I like real-time PCR now just as a last thing we’ll go over real quick how do we optimize and troubleshoot our real-time PCR reactions and optimization of real-time PCR reactions is important for this reason since the real-time PCR reactions are based on a doubling of product every cycle if the reaction is an optimize this doubling will not occur so to get precise calculations of quantity you need to have a doubling and to get an exact I bling that reaction has to be working well and a well optimized PCR reaction when spread out on a standard curve if you take your template and will have nice evenly spaced points on a standard curve and all the replicates will essentially be lines right on top of each other so at a hundred percent efficiency or a perfect doubling every reaction and fold serial dilutions will be spaced three point three cycles apart from each other and this this graph shows a really nicely optimized reaction you could get really good quantitative data from that top device PCR reactions people will nor normally design multiple primer sets and then empirically test each primers out with a standard curve don’t dilute out some template DNA a certain certain number of tenfold Aleutians for example and just see which primer set is working best then once you found the best primer set you can run a temperature gradient to find out the best kneeling temperature for those primers so it’s pretty straightforward and standard curves are ideal for assessing optimization and this isn’t required so much for or just demonstrating real-time PCR in the reaction or in the in the classroom but if you want to have reactions where you can do quantification accurate quantification it’s a good idea to optimize and least be able to explain to the students how to optimize PCR so troubleshooting why would it be important for teachers to be able to solve real-time pcr problems well one is it helps students have better success with our project so help reduce frustration preventing problems at the start can also help to avoid lost experiment and reagents and as we all know being able to explain unusual results leads to great teaching opportunities so this is kind of another teaching tip that I’ve noticed a lot of times students real-time PCR data isn’t perfect and that gives a great chance to explain to them how experimental design works how pipetting can be better how

how to mix reagents better things like that you can teach a lot based on problems that come up as the students are you’re doing their own real-time PCR if they’re doing their own projects for example so some basic troubleshooting a good real-time PCR reaction will have nice flat baselines their replicates for the same sample will be tightly clustered the lines will almost be on top of each other our dilution series will be spaced out evenly because it’s all about that left-right curve position that shows the quantity so if we make a dilution series a better be spaced out the curve should all be nicely s-shaped that’s the classic shape plateau height I alluded to this earlier but it really doesn’t matter PCR reactions just double and double and where they finally and it can vary over over a medium range so we don’t worry about the plateau height it doesn’t have to be the same the curve should be smooth and the melt curve should have only one product sometimes if things like this go wrong for example our replicas are tightly clustered might be pipetting error our PCR reactions may not be optimized samples might be evaporating because they weren’t sealed properly maybe we’ve got too much or too little over unknowns and it’s just too little to detect or maybe the instrument isn’t calibrated properly some instruments need celebration if our baselines aren’t flat like they should be that could also be sample evaporation because the plates were in sealed properly might be bubbles in the PCR reaction that are popping in the first few cycles or maybe the reagents weren’t properly mixed or if the software requires a baseline window to be set maybe that wasn’t set properly our dilution series may be messed up if there’s pipetting errors we may have the curves coming too close together or too far apart there may be too much DNA there may be PCR inhibitors maybe even too little DNA or the PCR reaction just isn’t that efficient and if the curves aren’t S shape sometimes curves can have really really strange shapes maybe it’s not actual PCR products at all maybe PCR didn’t even work and it’s just a sample that’s evaporating that you’re seeing pausing the fluorescence to change or it could be fluorescence drift and unamplified samples the cyber green may be bleaching out in any event there’s something seriously wrong in gas if you don’t see those sigmoid curves and if the curves aren’t smooth again it could be where pipetting or evaporation may be the assays got problems or maybe even there’s like electrical noise on the causing problems with the instrument melt curves if we have multiple Peaks in our melt curves that tells us that we’re amplifying more than one product might be normal for making primer dimers at very low concentrations but maybe it tells us that we’re using the wrong temperature for our primers and they’re binding and it kind of different site and making extra product it is a suggestion that primers need to be redesigned though if we start seeing multiple peaks and our melt curve so common themes that crop up here Karen pipetting careful in choosing the plastics and sealing the plates care needs to be taken an experimental design and proper use of positive and negative controls so this kind of goes into another teaching tip you can use real time PCR to teach the importance of proper designed experiments it’s a great learning experience to go through looking at real time PCR data I’m not knowing what’s going on with it because there weren’t positive and negative controls really kind of reinforces they need to have things like that in there and for any of these troubleshooting things our technical support group can can help you so you’re definitely not alone in resolving these issues so we’ve looked at these subjects today what is real-time PCR used for how does it work what chemicals and instruments are used to detect Kinane what is the real-time data look like how can we demonstrate it in the classroom and what are some ways to troubleshoot real-time PCR and with that I’ll turn it over to Ingrid for a moment thank you Dave before we answer questions I’d like to let you know that there are several resources available to you regarding real-time PCR these will be sent along to you with the playback of the webinar in a few days also we’d like to extend a special offer to each of you when you purchase the real-time instrument of your choice you can receive a free real-time PCR starter kit also of your choice that’s over a five hundred dollar

value please contact us at biotechnology underscore Explorer at to receive your quote for this special offer also at the end of this session you will have the opportunity to complete the exit survey to give us feedback on this session and to share other topics you’d like to have us present and now back to you Dave for questions thanks Ingrid okay so a bunch of questions have come in on good one is the animation of PCR available for download on the bio-rad site I’m pretty sure we do have that available right yeah yeah so we can we can put the link to that in the in the email that we send out after this another question that came in is is this the I cycler software that’s that was shown in here that software the screenshots I had were actually our CFX manager software that’s our newest one the eye cycler software is almost identical in terms of the workflow itself to set up the protocol on the plate and you still get the same looking result really any manufacturer’s real-time pcr is going to have that same basic workflow to it another question we had is is there a standard threshold or is it chosen so in that thought experiment I did the threshold was 1 million copies everything was based over when when those curves passed the 1 million point and what the software will generally do is it will pick a threshold point where all your curves are essentially parallel to each other so software will typically choose the threshold you can always choose your threshold manually and basically it’s either by using a standard curve or by just looking at the data III and looking at where your curves are parallel to each other let’s see oh this is a good question someone asked that they’re not clear on how a CT value can be anything other than a whole number because PCR is a set of discrete whole cycles and that that is true that the PCR cycles are discrete but what happens is the amplification curve being a logarithmic curve it could cross anywhere between individual cycles and it actually does that the curves are smooth curves so when when you connect the points on the curve those points will fall just anywhere between those those cycle numbers and you can actually tell the difference between a hoe typically around 0.1 CT units so a tenth of a CT or a tenth of a cycle you can reliably tell the difference in fervor a well optimized assing so it doesn’t have to be whole numbers but tenths of numbers is a the limit for the detection or the sensitivity on on telling one sample from another oh there’s a question about practical advantages of real-time PCR over conventional PCR and it depends on what the goal of the experiment is or the goal of the research so for example that the container ship full of GMO grains the the only way you can tell the percentage of GMO in that using PCR would be to use real-time PCR because of its quantitative nature if you used conventional PCR you’d certainly have a good chance of detecting genetically modified food in there but you never know if it’s above the 5% legal threshold or not the same with say forensics if you’re looking for just did a did a blood stain contain a DNA type that matched a suspect you could do that with conventional PCR but if someone was to turn around and ask well how much DNA was there was there really enough to accurately detect to do that one of the ways to do that would be real-time PCR no conventional PCR useful for anything where you want to tell what the sample is or what what the template was but real-time PCR if it’s needed for quantifying it or telling how much there was no there’s a question that came in about how many students would one typically have for the for the GMO experiment and when I run that kit in real-time in my class I would typically

have around 15 or so students I’d split them up into into pairs so we’d have maybe seven or eight work groups something like that if you end up with too many students and this is true for any teaching lab too many students and you can’t really give that much attention to the individual students that may not be following the whole thing so I’d say for that because of its complexity that’s that’s to keep it under 10 and work groups if you can just look at some other questions here oh okay someone asked you we have a recommended site for purchasing primers right now bio-rad doesn’t sell or make primers other than the ones that we supply in our kits already so places that we recommend people to get primers from would be something like IDT technologies or also bio search technologies both of those companies do a really good job at developing in that and selling primers opera and also fez primers to uh someone asked can we make a synthetic DNA from rt-pcr there’s actually two rt-pcr s I’m not sure which one that refers to one is real-time PCR and the other is reverse transcriptase PCR coming from messenger RNA sample either way the end product is DNA so no matter what you start off with you’re gonna end with DNA and that DNA can be put on an agarose gel it can be used in anything you’d use DNA from a conventional PCR reaction for someone asked is that the maximum curvature roach the maximum curvature approached that’s the kind of technical terminology that’s used to identify how a threshold is set for that bar that I had at the 1 million amplicon point when that’s automatically set in some software like RI cycler software that set on the maximum curve of the the maximum point of curvature on most of the traces so essentially it’s a fancy way of saying where the curves are all amplifying the most where that exponential increase is the most that’s the best place to put the threshold have a question can we record this presentation the presentation has been being recorded already and it will be available for download at some point after we’ll we’ll send out links after oh yeah like like this question how much background did the students need from someone who currently teaches ninth graders I have actually taught in in one of my PC our classes continuing ed students one of which had very little background definitely no college college background and I don’t think if I remember eight he had finished high school he by the end of it he was actually very comfortable with how DNA structure was how regular PCR worked and how real time PCR worked so even though a class may not understand all the concepts are all the levels I think a lot of these things are applicable to a wide eyed range and because real-time PCR touches on so many different things that structure of DNA how to draw graphs how to analyze graphs how to design experiments how chemistry works I think it can be used in a lot of different situations okay looks like we just have time for two more questions now so one question came in are all of the machines capable of precision melt analysis or do you need a special thermal cycler and the answer to that is that it it does take a specialized thermal cycler for precision melt regular melt analyses we can tell the difference between oh I don’t know the dozens of base pairs but if you’re trying to tell the difference in a single base pair you need a cycler that’s got a very precise block on it very precise temperature control and that can also be calibrated for any differences from one machine to the other and our CFX 96 system it can do precision melt analysis but our other systems like our mini opticon or a cycler series the those ones you wouldn’t be able to do the most precision of the precision belt analysis on and let’s see we had a question about bubbles in the wells how much of a real

issue is this it it varies I help people with real-time PCR everyday when they call in through tech support I’d say every two or three days I’ll encounter someone who sends in data where the problem was caused by by bubbles the easiest way to avoid that and you can use this for lots of things in your lab is if you do what’s called the reverse pipe heading where you pipette down to the second stop on your pipe patter to suck up three agents and then pipette down to the first stop it expel it into the tube and that way you’re not expelling a bubble in but anyone who wants more details on that they can just contact me after the fact and I can help them with that so I think that’s all the time we have questions for all the other questions we will answer and we’ll put in to put into the email that goes out after this and thanks everyone for attending there’s a lot of fun talking to you