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Gene expression analysis by Quantitative Real Time PCR - By using experimental example
 
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In this video I have explained that how we study relative gene expression by using quantitative Real Time PCR. To explain the concept real experimental data is used in this video. By watching this video you will learn following things - 1. Basic principle of quantitative real time PC 2. About SYBR Green and Taqman probe 3. How to study relative gene expression data by analyzing quantitative real time PCR data Please write to me in case of any question or doubt at - [email protected] Currently we are making a 60 Hrs video on complete cancer biology and also bigger video series on stem cell biology, immunology etc. For this cause we need your support, to donate please contact on whatsapp - +918698684792. For donation you can also contact on email - [email protected]
Views: 1883 Logical biology
The principle of Real Time PCR, Reverse Transcription, quantitative rt-PCR
 
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This video is an easy and full explanation about the principle of real time PCR. For better understanding watch the previous video about the principle of PCR: https://www.youtube.com/watch?v=Kx5qMjh-izA
How to Analyze Real-time PCR Data -- Ask TaqMan® Ep. 16 by Life Technologies
 
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Submit your real-time PCR questions at http://www.lifetechnologies.com/asktaqman In this video, Sr. Field Applications Specialist Doug Rains covers the various options that researchers have for performing final qPCR data calculations. Learn how to take advantage of Life Technologies' offerings of free external data analysis software, including DataAssist and ExpressionSuite. Welcome to AskTaqMan, where we answer your questions about real-time PCR. Here's an important question from Janaína at UFRGS in Brazil. She asks, "How do I analyze my Real-Time PCR data?" Well, there are in fact several options for analyzing data and generating final reports, depending on the particular application one is running. Let's address the most common qPCR experiment type: namely, gene expression. The first option is to have the instrument software perform calculations for you. In all of our most recent software versions, you have the option to set up new gene expression experiments by designating either comparative Ct or relative standard curve as your quantification method. You'll need to create at least 2 assay names -- one normalizer and one target -- and at least 2 sample names. You'll then assign these sample and assay names to the appropriate wells, making certain to identically label any wells representing pipetting replicates. If you're running the relative standard curve method, be sure to also label wells containing your dilution curve as standards, and to add standard amounts. There's a handy shortcut key that really helps set these up, by the way. Finally, tell the software which target on the plate is your normalizer gene and which sample you want to choose as the reference sample. - Most people choose the "untreated" sample, by the way. At the end of the run, simply go to either the Results or the Analysis tab, depending on your version, and to gene expression. If you labelled everything correctly, final fold change data will be generated for you at the end of the run and presented both in graphical and in tabular form. In the case of the latter, there's a column labelled RQ, or relative quantification, which is exactly the same as fold change. The instrument software has so many features for looking at your gene expression data that we just won't be able to go into too much detail in this video. So I suggest having a look at a copy of the Relative Quantification Getting Started Guide. It's available as a hard copy, as well as electronically online. And if you click on your software's Help menu, you'll even find a version right at your fingertips. But what about other options? In fact, Life Technologies offers two other exceptional tools for calculating gene expression data. Both are fre-standing packages that can be downloaded and used at no charge from the Life Technoligies website. Both DataAssist and Expression Suite offer intuitive workspaces and plenty of data crunching capabilities. Both can do multi-plate studies, calculate biological replicate fold change data, and present results in a variety of forms, including heat maps, volcano and scatter plots, and more. If you'd like to learn more about either of these packages, there are free video tutorials available on the web. Just go to learn.lifetechnologies.com, click on Gene Expression, and twirl down to the section on Web-Based training.
AriaMx: Analyzing a Quantitative PCR Experiment
 
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The AriaMx Real-Time PCR System is a fully integrated quantitative PCR amplification, detection, and data analysis system. The system design combines a state-of-the-art thermal cycler, an advanced optical system with an LED excitation source, and complete data analysis software. Updated August 2016. More information at http://www.genomics.agilent.com/campaign.jsp?id=4600006&cid=G011543
Views: 10752 Agilent Technologies
Finding Multiple Melt-Curve Peaks When Using SYBR® Green in Real-Time PCR  -- Ask TaqMan®: Ep. 8
 
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Submit your Real-Time PCR questions and watch the rest of our videos at http://ow.ly/bQh0l. Life Technologies Sr. Field Application Specialist Doug Rains helps with the understanding the causes of multiple peaks in a melt curve when using SYBR® Green dye in Real-Time PCR. SYBR® Green I chemistry is a free-floating dye. Here's how it works. When SYBR® Green dye is just swimming around in the tube, it doesn't give off much fluorescence -- even when we zap it with the light source on a real-time PCR machine. But SYBR® Green dye really likes to bind to double-stranded DNA. And, when it does, and you hit it with light, the dye gets excited and fluoresces. In theory, the basic idea, then, is this: as PCR creates more and more product, the signal of SYBR® Green dye should go up proportionally. In practice, this doesn't always happen. That's because SYBR® Green dye binds to any double-stranded DNA. Meaning, every double-stranded molecule in the tube will bind SYBR® Green dye and add to the fluorescent signal. Because of this concern, users run melt curves after each experiment. They do this by slowly raising the block temperature from about 60 degrees Celsius up to 95 degrees Celsius and monitoring fluorescence. As you can see, signal drops slowly until at some point, it drops off suddenly to zero. Halfway down this drop-off is the presumed melting temperature of the product created during PCR. If you have the software do a little calculus for you, you get what's called the derivative view, which I find a little more helpful, since the drop-off gets converted into a peak. What you're hoping to see is one, clearly defined peak, which suggests— doesn't prove, mind you— but suggests that you got clean amplification of a single product. One thing you don't want to see is multiple peaks, as this suggests your amplification curves are a composite of more than one product. So what causes extraneous peaks? It really depends. It could be non-specific amplification or primer-dimer formation. In the first case, you'll need to redesign your primers to a more specific sequence. In the latter case, you may just need to lower the concentration of primer to discourage dimer formation, although a primer redesign may ultimately be necessary. The problem is, it's difficult to know exactly what's causing certain anomalies, so some users end up spending a lot of time repeating failed experiments under multiple new conditions or with multiple new primer sets. That's neither fun nor affordable. Still, SYBR® chemistry is perfectly valid for qPCR when all things go well. Users just need to take more care than users of TaqMan® chemistry do in designing their experiments, and take additional quality control steps when evaluating their data.
Analyzing Quantitative PCR Data
 
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Relative and absolute methods of qPCR analysis. Created for an assignment for BIOC3001: Molecular Biology at the University of Western Australia. ****SCRIPT**** [I know it's a bit fast] qPCR or quantitative real-time PCR… ….is simply classic PCR monitored using fluorescent dyes or probes. qPCR is accurate, reliable and extremely sensitive, it can even detect a SINGLE copy of a specific transcript. qPCR is commonly coupled to reverse transcription to measure gene expression. No wonder it is so important for molecular diagnostics, life sciences, agriculture, and medicine. Firstly, let's go over the NUTS and BOLTS of qPCR. For this you use a fluorescent dye which binds to the DNA. As qPCR progresses, the fluorescent signal increases. Ideally the signal should double with every cycle, which is then plotted. Because there are few template strands to start with, initially there’s a faint signal. Eventually, usually after 15 cycles, the signal rises above the background noise and can be detected. We call this the THRESHOLD CYCLE, Ct, the point from which all quantitative data analysis begins. But how do you analyse qPCR data? You can either use an absolute quantification method, with a standard curve, OR a relative method, using one or more reference genes to standardize and compare the differences in Ct values between two treatments. The absolute standard curve method determines ORIGINAL DNA concentration by comparing the Ct value of the sample of interest with a standard curve. To create the standard curve, you need to make DNA samples of different KNOWN concentrations. After doing PCR on these, you will see different PCR plots for each standard ….. and unsurprisingly they have different Ct values. The GREATER the concentration of the original DNA sample, the SMALLER the Ct value. So if you plot ORIGINAL DNA concentration against the Ct values. You will have a standard curve like this….. Now let’s say the PCR plot of your unknown DNA sample is somewhere here….. ...which corresponds to this Ct value on the standard curve here…. Using the standard curve you can figure out the log concentration of your DNA sample to be x. As this is in log scale, you can simply calculate your sample DNA concentration to be 10 to the power of x. Absolute analysis is suitable when you want to determine the ACTUAL transcript copy number, that is the level of gene expression. On the other hand, Relative quantification is used when you want to COMPARE the difference in gene expression BETWEEN two treatments, for example light or dark treated Arabadopsis thaliana. This is done using one or more reference genes, such as actin, which are expressed at the SAME level for both treatments. You then perform qPCR on both your samples and the reference genes, find out the DIFFERENCE between the two Cts values, delta Ct, in EACH treatment. Now the RATIO of the two delta Cts …[pause a bit] . tells you how much gene expression has changed. For instance, in the dark treatment, the Ct value of your reference gene is at THIS level, the Ct value of your target gene is THIS Level. So you have this delta Ct which is the difference in Cts in the first treatment. in the dark treatment, the Ct value of your reference gene is STILL at THIS level, but the Ct value of your target gene may become only this much. So the ratio of the two Ct values is.. delta Ct(dark treatment) divided by delta Ct(light treament) equals one third ….showing the delta Ct has DECREASED by a factor of 3, which means that gene expression of the target gene is GREATER in the dark treated sample. This is how relative quantification using a reference gene helps detect change in the expression of your target gene. In conclusion, there are two ways to quantify transcripts using qPCR: absolute quantification using a standard curve, and relative quantification using a reference gene. The method used depends on whether you want to determine the ACTUAL number of transcripts or the RELATIVE change in gene expression.
Views: 203275 TARDIStennant
Real-Time Polymerase Chain Reaction (PCR) - Multi-Lingual Captions
 
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This short animation introduces the real-time polymerase chain reaction (PCR) procedure. Captions are available multiple languages. This resource was developed by Yaw Adu-Sarkodie of the Kwame Nkrumah University of Science and Technology and Cary Engleberg of the University of Michigan. It is part of a larger learning module about laboratory methods for clinical microbiology. The full learning module, editable animation, and video transcript are available at http://open.umich.edu/education/med/oernetwork/med/microbiology/clinical-microbio-lab/2009). Copyright 2009-2010, Kwame Nkrumah University of Science and Technology and Cary Engleberg. This is licensed under a Creative Commons Attribution Noncommercial 3.0 License http://creativecommons.org/licenses/by-nc/3.0/. Help us caption & translate this video: http://amara.org/v/BVm4/
Views: 594202 openmichigan
Real Time QPCR Data Analysis Tutorial
 
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In this Bio-Rad Laboratories Real Time Quantitative PCR tutorial (part 1 of 2), you will learn how to analyze your data using both absolute and relative quantitative methods. The tutorial also includes a great explanation of the differences between Livak, delta CT and the Pfaffl methods of analyzing your results. For more videos visit http://www.americanbiotechnologist.com
Views: 352917 americanbiotech
Baselines in Real-Time PCR -- Ask TaqMan®: Ep. 5
 
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Submit your Real-Time PCR questions and watch the rest of our videos at http://ow.ly/bQh0l. Life Technologies Sr. Field Application Specialist Doug Rains helps with the understanding of baselines in Real-Time PCR. We're looking at a fairly standard real-time amplification plot. We have some nice curves, each of which has the familiar geometric phase, linear phase, and plateau phase. So far, so good. But what's all this . . . junk in the early cycles? Well, friends, if you said "junk," you were right. That's right, I said it -- junk, trash, waste, detritus, garbage, otherwise known as noise. It's the stuff we see before our actual signal from amplification gets high enough to overcome that noise. And, as the rather impolite adjectives I used a second ago would suggest, it's completely useless to us. This noise does have an effect on our curves. Our job is to minimize that effect by effectively subtracting out the noise. We do that by establishing what's known as a baseline -- a cycle-to-cycle range over which only noise can be seen, prior to the appearance of curves. Once established, the software will effectively subtract out the noise on a well-by-well basis, greatly improving the quality of our data. Let's switch the Y-axis to linear scale for a moment to illustrate the effect of baseline subtraction. Here's our data prior to baselining. Note how every sample begins from a slightly different spot on the Y-axis, causing our geometric phase data— this curvy part over here when we're in a linear scale— to look horrible. But once we subtract noise, every sample begins from the same point 0. And as a result, the data clean up nicely. The value we get after normalizing for background noise is something called delta-Rn. If you ever look closely at a log-scale amplification curve— the one we're used to seeing— you'll notice that delta-Rn is what's graphed on the Y-axis. But before you go, just note that there are two ways to set baselines in Applied Biosystems® real-time PCR software: manually, and automatically. If you do it the manual way, you set the baseline range under Analysis Settings. You either set it for a single assay, in which case all wells for that assay get the same subtraction . . . or you can go under Advanced Settings and set wells individually. Better yet, just use the default setting of Auto Baselining. With this selected, the software figures out how much noise needs to be subtracted from each well individually, and, as such, generally produces the best results. So why have a manual feature? Well, Auto does fail on occasion, especially with some SYBR® Assays and non-standard chemistries. You'll know auto has malfunctioned by the shapes of your curves. If they look more S-shaped than they should, it could be that auto has misapplied the baseline and set the End cycle too low. As a result, not enough noise is being subtracted, and the curves take on a strange shape. To fix the problem, switch over to manual mode for that assay and raise the End cycle until the curves take on a regular shape.
Real time PCR
 
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This pcr reaction lecture explains about real time pcr procedure. It explains the realtime pcr mechanism and uses in molecular diagnosis. Web-http://shomusbiology.com/ Download the study materials here- http://shomusbiology.weebly.com/bio-materials.html A quantitative polymerase chain reaction (qPCR), also called real-time polymerase chain reaction, is a laboratory technique of molecular biology based on the polymerase chain reaction (PCR), which is used to amplify and simultaneously quantify a targeted DNA molecule. For one or more specific sequences in a DNA sample, quantitative PCR enables both detection and quantification. The quantity can be either an absolute number of copies or a relative amount when normalized to DNA input or additional normalizing genes. The procedure follows the general principle of polymerase chain reaction; its key feature is that the amplified DNA is detected as the reaction progresses in "real time". This is a new approach compared to standard PCR, where the product of the reaction is detected at its end. Two common methods for the detection of products in quantitative PCR are: (1) non-specific fluorescent dyes that intercalate with any double-stranded DNA, and (2) sequence-specific DNA probes consisting of oligonucleotides that are labelled with a fluorescent reporter which permits detection only after hybridization of the probe with its complementary sequence to quantify messenger RNA (mRNA) and non-coding RNA in cells or tissues. qPCR is the abbreviation used for quantitative PCR (real-time PCR).[1] Real-time reverse-transcription PCR is often denoted as: qRT-PCR[2][3][4] The acronym "RT-PCR" commonly denotes reverse transcription polymerase chain reaction and not real-time PCR, but not all authors adhere to this convention. Source of the article published in description is Wikipedia. I am sharing their material. Copyright by original content developers of Wikipedia. Link- http://en.wikipedia.org/wiki/Main_Page
Views: 221777 Shomu's Biology
Melt curve analysis in qPCR experiments
 
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The video was created by students as part of an assignment in Biochemistry (BIOC3001) in the School Molecular Sciences (Biochemistry and Molecular Biology) at the University of Western Australia. If you would like to use this video for teaching, please acknowledge: the 'BIOC3001 students at the University of Western Australia, School of Molecular Sciences'.
RT-PCR for Gene Expression
 
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RT-PCR for Gene Expression
Views: 45087 Matthew Bremgartner
7_Quantitative PCR -- the melting curve
 
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What exactly is provided by the curve displayed at the end of my PCR run? What is "-d(RFU)/dT"? How do I obtain a melting temperature from this curve, and what does it tell me about the amplification of primer dimers?
Views: 23092 Matthias Dobbelstein
Real Time PCR - Interpretation of the amplification plot - part 2 HD
 
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This tutorial will discuss the basics of how to interpret an amplification plot of real time PCR. introduction Real Time PCR: http://youtu.be/EaGH1eKfvC0
Views: 44189 MrSimpleScience
Real Time PCR Video 1 RNA Analyses and Why Real-time PCR?
 
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In this first video, the basic concept of why using real-time PCR for gene expression study is discussed. This may be an old-school method, but since this is so powerful and useful, many scientists are still using real-time PCR to do gene expression study as of today.
Views: 163 King Ming Chan
Real-time PCR
 
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( http://www.abnova.com ) - Real-time PCR, also called quantitative real time PCR (Q-PCR/qPCR), is used to amplify and simultaneously quantify a targeted DNA molecule. It enables both detection and quantification (as absolute number of copies or relative amount when normalized to DNA input or additional normalizing genes) of one or more specific sequences in a DNA sample. More videos at Abnova http://www.abnova.com
Views: 242040 Abnova
Using Standard Curve to Estimate DNA Quantity - Forensic Focus #4
 
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Submit your questions at http://www.thermofisher.com/forensicfocus Everyone wants to know how much DNA is in their extract, but then they ask: how can I tell if my estimate is accurate? The standard curve holds the answers. A standard curve is a tool that allows us to estimate the DNA concentration of unknown samples by comparing them to standards with known DNA concentrations. In this example, the standards consist of a 10-fold dilution series ranging from 50 ng/ul down to 5 pg/ul. During each PCR cycle, the amount of fluorescent signal for each standard in the dilution seies is measured. When the fluorescent signal crosses the detection threshold the cycle number is recorded as a Ct value, or threshold cycle value. The Ct value is what ultimately is used to create the standard curve. The Ct values are inversely proportional to the concentration of DNA in the standards. The high-concentration, 50 ng/ul standard will cross the detection threshold first, generating a “low” Ct. The low-concentration, 5 pg/ul standard will take many more cycles to cross the same threshold - and therefore the Ct will be higher. The Ct values for each dilution of the standard curve are plotted on a graph, and the software generates a regression line that fits the data. Because the standards are 10-fold dilutions, we expect the change in Ct from one standard to the next to be uniform. An uneven distribution of Ct values might indicate that the dilution series was not accurately pipetted. Let’s take a look at the standard curve for a specific DNA target, the small autosomal target. The X axis is the log of the known standard concentrations. The Y axis is the Ct value of each standard. Now, Do you see the quality metrics at the bottom of the screen? Let’s review Slope, Y intercept, and R2.? The slope measures the efficiency of the PCR reaction. In a perfect world, a slope of -3.3 indicates that the PCR reaction is 100% efficient; the target DNA is doubled each cycle. Two copies become four; four become eight; and so on. The Y intercept is the expected Ct value for a 1ng/ul sample. The R2 value measures how well the regression line fits the data points. A line that fits the data points perfectly has an R2 of 1. If your data points are scattered, the R2 value for the line will be lower. The Ct values of your standards affect the slope, the Y intercept, and the R2 value. It is very important to prepare the standard dilution series carefully to ensure consistent and accurate results! Running the standards in duplicate can help ensure you have a high quality standard curve. Once your standard curve passes the metrics test, it can be used to evaluate an unknown sample! The Ct value of the unknown sample is measured, and compared to the standard curve to estimate the DNA concentration of the unknown sample. Couldn’t be simpler! That’s it for today. If you have other questions, just click on the link below. And don’t forget- when in doubt, refer Back to the Bases!
rtPCR animation
 
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RT PCR animation - This lecture explains about the RT PCR also known as the real time PCR. Realtime PCR is a technique of amplifying DNA fragments with polymerase chain reaction and along with the PCR. it can help us to monitor the concentration of amplified DNA Replication in real time with the help of fluorescence emission. Real time PCR or RT PCR uses fluorescence resonance energy transfer or Fret to detect the fluorescence generated from the DNA amplification. Animation source is - Sumanas Inc. www.sumanasinc.com For more information, log on to- http://www.shomusbiology.com/ Get Shomu's Biology DVD set here- http://www.shomusbiology.com/dvd-store/ Download the study materials here- http://shomusbiology.com/bio-materials.html Remember Shomu’s Biology is created to spread the knowledge of life science and biology by sharing all this free biology lectures video and animation presented by Suman Bhattacharjee in YouTube. All these tutorials are brought to you for free. Please subscribe to our channel so that we can grow together. You can check for any of the following services from Shomu’s Biology- Buy Shomu’s Biology lecture DVD set- www.shomusbiology.com/dvd-store Shomu’s Biology assignment services – www.shomusbiology.com/assignment -help Join Online coaching for CSIR NET exam – www.shomusbiology.com/net-coaching We are social. Find us on different sites here- Our Website – www.shomusbiology.com Facebook page- https://www.facebook.com/ShomusBiology/ Twitter - https://twitter.com/shomusbiology SlideShare- www.slideshare.net/shomusbiology Google plus- https://plus.google.com/113648584982732129198 LinkedIn - https://www.linkedin.com/in/suman-bhattacharjee-2a051661 Youtube- https://www.youtube.com/user/TheFunsuman Thank you for watching the tutorial on RT PCR animation.
Views: 163002 Shomu's Biology
Quantitative real time PCR (qPCR)
 
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Lecture on quantitative real time pcr or qpcr to understand gene amplification in realtime. http://shomusbiology.com/ Download the study materials here- http://shomusbiology.weebly.com/bio-materials.html A quantitative polymerase chain reaction (qPCR), also called real-time polymerase chain reaction, is a laboratory technique of molecular biology based on the polymerase chain reaction (PCR), which is used to amplify and simultaneously quantify a targeted DNA molecule. For one or more specific sequences in a DNA sample, quantitative PCR enables both detection and quantification. The quantity can be either an absolute number of copies or a relative amount when normalized to DNA input or additional normalizing genes. The procedure follows the general principle of polymerase chain reaction; its key feature is that the amplified DNA is detected as the reaction progresses in "real time". This is a new approach compared to standard PCR, where the product of the reaction is detected at its end. Two common methods for the detection of products in quantitative PCR are: (1) non-specific fluorescent dyes that intercalate with any double-stranded DNA, and (2) sequence-specific DNA probes consisting of oligonucleotides that are labelled with a fluorescent reporter which permits detection only after hybridization of the probe with its complementary sequence to quantify messenger RNA (mRNA) and non-coding RNA in cells or tissues. qPCR is the abbreviation used for quantitative PCR (real-time PCR).[1] Real-time reverse-transcription PCR is often denoted as: qRT-PCR[2][3][4] The acronym "RT-PCR" commonly denotes reverse transcription polymerase chain reaction and not real-time PCR, but not all authors adhere to this convention. Source of the article published in description is Wikipedia. I am sharing their material. Copyright by original content developers of Wikipedia. Link- http://en.wikipedia.org/wiki/Main_Page
Views: 97270 Shomu's Biology
Fixing Software Setup Mistakes in Real-Time PCR (StepOnePlus™) -- Ask TaqMan®: Ep. 10
 
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Submit your Real-Time PCR questions and watch the rest of our videos at http://ow.ly/bQh0l. Life Technologies Sr. Field Application Specialist Doug Rains offers advices for fixing common software set-up mistakes when performing Real-Time PCR. I'm showing you a completed run file. Let's say I made a lot more mistakes than you did while setting up my file: wrong sample names, wrong dyes, and, yes, even forgot to label some wells. As you can see, I'm in Analysis under the Experiment Menu. To fix things, I'm going to go up here and click Setup. In the Plate Setup window, you can see my list of targets and samples. If I originally entered information incorrectly, I can change it right here. Let's say it's something as simple as a sample name. I activate the offending box, type the new name, and hit Enter. I now go to my plate map, which I access by clicking this tab, and I find that the new sample name has been added to the appropriate wells automatically. Okay, let's go back to Define Targets and Samples. Now what if I assigned the wrong fluorescent label to one of my assays? This one says FAM, but it should say VIC. That mistake left unfixed will definitely cause some analysis issues. However, I can just use the Reporter pull-down menu and make the switch. When I now go back to Analysis and click the analyze button, my change gets applied. And of course, the data improve dramatically. How is this possible? It's possible because whenever the instrument takes readings, it does so through every filter set, regardless of your dye assignment. Thus, the raw data are always there in the file. Back to Setup, where we'll deal with the issue of missing well assignments. Row D is blank because somebody forgot to assign assays and samples. And so these wells yield no data. But if I simply make the assignments now like so then go back to Analysis and reanalyzed the data, curves for those wells will appear. So, what information can we change after the fact? Just about anything besides cycling conditions. That includes sample and Assay names, Tasks (such as which wells are standards), standard amounts, the passive reference dye, and plenty more. Not only that, every Life Technologies real-time PCR instrument gives you this leeway. So even if you're using an older instrument, that's okay. In fact, you can even change the experiment type on all of the newer Life Technologies platforms, So if you accidentally labeled your ddCt run as a standard curve experiment, you can change that.
How To Perform The Delta-Delta Ct Method (In Excel)
 
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In this video tutorial, I will show you how to perform the delta-delta Ct method by using Microsoft Excel. The delta-delta Ct method is a simple formula used in order to calculate the relative fold gene expression of samples when performing real-time polymerase chain reaction (also known as qPCR). THE ONLINE GUIDE http://toptipbio.com/delta-delta-ct-pcr/ MASTERING QPCR - THE ONLINE COURSE https://bit.ly/2CIVBMq VIDEO BREAKDOWN Step 1: Average the Ct values (00:53) Step 2: Calculate delta Ct (01:59) Step 3: Calculate the average delta Ct for controls (02:46) Step 4: Calculate delta-delta Ct (03:39) Step 5: Calculate 2^-(delta-delta Ct) (04:54) MORE HELPFUL HINTS & TIPS http://toptipbio.com/ FOLLOW US Facebook: https://www.facebook.com/TopTipBio/ Twitter: https://twitter.com/TopTipBio LinkedIn: https://www.linkedin.com/company/top-tip-bio/
Views: 37274 Top Tip Bio
Soya Analysis: Real-time PCR Sample Preparation
 
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How to prepare processed samples for real-time PCR using SureFood® PREP Advanced
Views: 2757 R-Biopharm AG
Real-time PCR Applications -- Ask TaqMan® Ep. 17 by Life Technologies
 
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Submit your real-time PCR questions at http://www.lifetechnologies.com/asktaqman In this video, Sr. Field Applications Specialist Doug Rains covers several possible applications that are possible on Life Technologies real-time PCR instruments. Learn about both quantitative and non-quantitative applications, including those that interrogate DNA, RNA and even protein. Finally, discover convenient options for acquiring high-quality TaqMan® assays for all of these experiment types. Bon giorno, e benvenuti a Ask TaqMan. I'd like to read a question I received recently from Giampiero at the University of Pisa. He asks, "What are the most common applications for real-time PCR?" Well, most notable on the list is looking at relative expression levels of gene targets. Users compare two or more samples -- say, cells from untreated and treated animals -- in order to determine if and how much the expression of their gene of interest is changing. Using Life Technologies real-time systems, in combination with either custom or pre-designed assays, users can look at either messenger RNA or small RNA targets, including specific micro RNAs. So what else can a real-time user do with her instrument? So many applications, so little time. A common one is using a standard curve to calculate absolute or relative copy number of a DNA target. This could be a pathogen in food or animal samples, a microbe in water, or any other detectable nucleic acid sequence. Speaking of food, some labs use qPCR to calculate %GMO in food samples in order to ensure that they meet legal standards. Copy number studies on genomic DNA targets are quite important to any number of labs, who typically need either to segregate animals with different copy numbers of a transgene, or to detect and quantify duplication or deletion events associated with a particular phenotype. Keep in mind, I only mention some of the more common possibilities. Fact is, pretty much any application that requires quantifying nucleic acid targets is possible in real-time. So what about experiments that aren't quantitative? The real-time system has you covered there, as well. Perhaps most notable is the allelic discrimination assay, which detects specific single nucleotide polymorphisms in DNA samples, then segregates samples based on their homozygous or heterozygous genotyopes. For researchers wanting to merely detect rather than quantify pathogens, including those present in samples at extremely low levels, our presence/absence application and associated detection reagents are an excellent option. There's also an option on many Life Technologies instruments to run high-resolution melting experiments, which can be used to check percent-methylation of short DNA target regions or to discover small sequence differences among multiple samples. Besides nucleic acid-based work, real-time is increasingly being used to look at proteins. Two applications worth mentioning are Life Technologies Protein Expression technology, which lets users determine relative expression levels of specific protein targets, as well as our protein thermal shift assay, the perfect application for screening different buffer conditions and ligands for their effect on protein thermal stability. And don't forget: regardless of the application, Life Technologies offers a full suite of reagents, assays, and analysis software to help you get the job done. That of course includes millions of pre-developed TaqMan Assays for a number of applications, including gene expression, SNP genotyping, and many more.
What is Real Time PCR?
 
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Learn more about Real Time PCR in this video clip. For the full webinar please visit: http://bitesizebio.com/webinar/28767/qpcr-tips-workflow-applications-and-troubleshooting/
Views: 4030 Bitesize Bio
How to Normalize cDNA Concentrations -- Ask TaqMan® Ep. 15 by Life Technologies
 
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Submit your real-time PCR questions at http://www.lifetechnologies.com/asktaqman In this video, Sr. Field Applications Specialist Doug Rains examines why the choice of an appropriate normalizer gene is so critical for the accuracy of final real-time PCR data. In addition to discussing this gene's importance, Doug provides a helpful reference document which explains in detail how researchers can quantitatively validate their choice of a control gene. Relative gene expression is one of the most common applications that researchers perform on their real-time instruments. So it's never a surprise when I receive a question like the one I got recently from Shan at Pennsylvania State University, who asks the following:, "Since every cDNA is run with both the gene of interest and internal control, do I have to be sure that the concentration of each cDNA is the same? Excellent question. Let's start with a little bit of review. Whenever we do a gene expression experiment, we need at least two gene-specific assays one for our gene of interest, and one for an internal control gene, also sometimes referred to as a "normalizer," "housekeeping gene," or "endogenous control." Different terms, same idea. So what does this second gene -- the normalizer -- do for us? Essentially, its number one job is to normalize our final data for differing input amounts of template. Here's what I mean. Say I'm comparing the expression of my gene of interest in two samples: an untreated and a treated cell line. When I examine the real-time results, I find that my two samples differ by a single Ct. This result suggests that there's a two-fold difference in my target's expression between the two samples. But how do I know that two-field difference is real? After all, isn't it possible that my untreated and treated cDNAs had different concentrations, and that's the reason we're seeing a one-Ct difference? Definitely a possibility. Precisely why I also have to run a normalizer gene on each sample. A good normalizer gene is one whose expression is stable across my various sample types, assuming equal starting amounts. Thanks to the data I collect from this second gene assay, I can effectively monitor input amounts of cDNA, even when they differ from sample to sample. Then, at the end of the run, I can use Ct data from the normalizer gene to correct final expression values for differing input amounts of cDNA. I can thereby avoid having to always add equal amounts of template when running my experiments. But clearly, for the normalizer to do its job correctly, its expression has to be stable across our sample set. If it's not, the data from the normalizer can actually make our final expression values less accurate. So how does one choose a good normalizer? To find out, I strongly recommend our kind viewers visit the Life Technologies web page, where you can download a copy of the Relative Gene Expression Workflow document. This PDF has an entire section on choosing the most appropriate normalizer, based not on guesswork, but on good, hard empirical data.
Real Time QPCR Data Analysis Tutorial (part 2)
 
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In this Bio-Rad Laboratories Real Time Quantitative PCR tutorial (part 2 of 2), you will learn how to analyze your data using both absolute and relative quantitative methods. The tutorial also includes a great explanation of the differences between Livak, delta CT and the Pfaffl methods of analyzing your results. For more videos visit http://www.americanbiotechnologist.com
Views: 174078 americanbiotech
High Resolution Melt Analysis Tutorial
 
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High resolution melt (HRM) analysis is a relatively new technique used in detecting small variations in DNA sequences between varying populations. Important applications of HRM include SNP analysis, genotyping and methylation analysis. In the following 20 minute tutorial presented by Sean Taylor, Field Application Specialist, Bio-Rad Laboratories, you will learn the basics of high resolution melt analysis and how to practically use it in your research.
Views: 28485 americanbiotech
Troubleshooting qPCR - What are my amplification curves telling me?
 
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Quantitative PCR (qPCR) is the method of choice for accurate estimation of gene expression. Part of its appeal for researchers comes from having a protocol that is easy to execute. However when your reactions do not result in ideal amplification, troubleshooting "why" can be challenging. Factors including sample quality, template quantity, master mix differences, assay design, and incorrect primer or probe resuspension can all influence efficient amplification. When troubleshooting, analysis of the appearance of your amplification curve can give you clues towards improving your results. This webinar will present a variety of problematic qPCR issues and how they are manifested in the amplification curve.
Understanding Melt Curves for Improved SYBR® Green Assay Analysis and Troubleshooting
 
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qPCR assays using intercalating dyes, such as SYBR® Green dye, are an economical and effective tool for measuring gene expression. To interpret intercalating dye assays, users need to know how to analyze melt curves, and understand the benefits and limitations of melt curve analysis. In this presentation, Nick Downey, PhD, covers melt curve basics and shares examples of multiple peaks due to suboptimal sample prep, primer dimers, and asymmetric GC content of amplicons. He demonstrates troubleshooting strategies. Experienced and novice users will benefit from an overview of uMelt software, developed by the Wittwer lab at the University of Utah, that can predict the melt profile of your assay before you run your experiment.
ClinilabTube: QIAGEN's real-time PCR system (Rotor-Gene Q)
 
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Rotor-Gene Q - Germany REAL-TIME PCR system from Clinilab - Egypt شركة كلينيلاب Cell phone : 0106 469 4374 Fax : 02-25257210 The Rotor-Gene Q is an innovative real-time cycler that enables high-precision real-time PCR thanks to its unique rotary design. The tubes rotate rapidly in a chamber of moving air, which results in uniform and accurate temperatures for every sample. When each tube aligns with the detection optics, the sample is illuminated and the fluorescent signal is rapidly collected by a single, short optical pathway. This unique design results in sensitive and fast real-time PCR and eliminates the sample-to-sample variations that typically occur in block-based instruments. Interchangeable rotors provide the flexibility to use different sample volumes and tube formats. Advanced instrument design also enables superior performance in High Resolution Melt (HRM) analysis. All state-of-the-art PCR and HRM analysis procedures are supported by a comprehensive software package. To take full advantage of this advanced cycling technology, use the Rotor-Gene Q in combination with specially optimized QIAGEN PCR kits and assays. The Rotor-Gene Q provides: ************************* * Outstanding thermal and optical performance * More applications than any other real-time cycler * Unmatched optical range with up to 6 channels spanning UV to infra-red wavelengths * Robust design for easy setup and minimal maintenance * Full compatibility with the QIAgility for automated PCR setup join Clinilab on Facebook : https://www.facebook.com/clinilab.analysis
How to Optimize qPCR using SYBR Green Assays - Ask TaqMan #38
 
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Learn More: http://www.thermofisher.com/sybr Are you new to using SYBR Green Assays for qPCR or having trouble getting accurate results? Today, let’s discuss how you can design and optimize qPCR using SYBR Green assays as the detection method. First, beware of reverse transcription (RT) bias when converting RNA to cDNA. Nearly all RT enzymes have the potential to introduce RT bias. When this happens the amount of cDNA won’t align with the amount in RNA samples. Learn More about RT Methods in this video: https://www.youtube.com/watch?v=Y-8OuXFFJz0 You can test for RT bias by reverse transcribing two-fold dilutions of a known amount of RNA. Then run a qPCR standard curve for each assay and endogenous control. The standard cure should be linear with a target slope of -3.323.” Once the cDNA is generated make sure to use the right primers for the qPCR. You will need to use some bioinformatics to design your primers, such as a tool like SNPMasker. In general, primers should be 20 nucleotides in length with a GC content in the 30-70% range. The last 5 nucleotides at the 3’ end should include no more than two G or C bases to avoid specificity issues. Finally, amplicons should be short -- generally between 50 – 150 base pairs. The next step is primer validation. The objective is to find the right concentration of forward and reverse primers that will yield the most robust assay without non-specific amplification or primer dimers. This is accomplished by running multiple qPCRs with 3 different concentrations of forward and reverse primers in a matrix format. The appropriate range of primer concentration is determined by the master mix. For instance, Applied Biosystems PowerUp SYBR Green Master Mix works best with primer concentrations in the range of 300 – 800 nM. Getting back to our experiments to optimize primer concentrations, the next step is to evaluate the Ct and run a melting curve also known as a dissociation curve for each primer concentration combination. If dissociation curve shows primer-dimers, there are two options: A. Start over and redesign the primers. B. Alter cycling temperatures to remove primer-dimers. The last step in ensuring that your primer set is going to yield usable, reproducible data is to ensure the PCR efficiency is within 90 – 110%. You can do this by simply running a standard curve with at least 5 logs of input DNA and using the software on your instrument to calculate PCR efficiency. If this all seems too complicated, you can use pre-designed TaqMan assays instead, which removes the primer design variable and ensures the best possible primer set Once the primers are designed, experimental analysis can begin. Here’s a tip! For measuring relative gene expression levels of two different samples, most researchers use what’s known as the ΔΔCt (ddCt) method. This analysis generates relative changes form one state to the next, much like a disease state versus treatments For more information about SYBR Green experiments, TaqMan assays or related reagents, please visit http://www.thermofisher.com/qpcr or http://www.thermofisher.com/sybr And If you have more questions concerning SYBR green assays, ΔΔCt analysis or any other qPCR questions, remember to Ask TaqMan and submit your questions on our website http://www.thermofisher.com/ask Thanks for watching!
qPCR Analysis with CFX Maestro™: Data Analysis
 
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This tutorial will explain how to perform data analysis and statistical analysis of your qPCR experimental results in the CFX Maestro Software. Covered topics include: - Viewing amplification data - Analyzing standard curves - Performing data QC - Gene expression analysis - P-values and performing ANOVA CFX Maestro Software provides detailed qPCR analysis including: - Gene Expression analysis - Multiple data visualization modes - Statistical analysis with t-tests and ANOVA - High-resolution graph export CFX Maestro Software is also available in a Security Edition for 21CFR part 11 needs, and in a Mac version for data analysis. We Are Bio-Rad. Our mission: To provide useful, high-quality products and services that advance scientific discovery and improve healthcare. At Bio-Rad, we are united behind this effort. These two objectives are the driving force behind every decision we make, from developing innovative ideas to building global solutions that help solve our customers' greatest challenges. Connect with Bio-Rad Online: Website: http://www.bio-rad.com/ LinkedIn: https://www.linkedin.com/company/1613226/ Facebook: https://www.facebook.com/biorad/ Twitter: https://twitter.com/BioRadLifeSci Instagram: @BioRadLabs Snapchat: @BioRadLabs
Views: 7038 Bio-Rad Laboratories
Understanding Reverse Transcriptase – Effects on Ct value
 
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The reverse transcription step is one of the greatest sources of variation in RT-qPCR. With SuperScript IV reverse transcriptase, the Ct value can be reduced by as much as 8. This enzyme outperforms wild type reverse transcriptases with better sensitivity at lower target concentrations. And it shortens the reaction time to just 10 minutes.
Automated systems for real-time PCR analysis
 
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Automated systems for real-time PCR analysis The challenge of achieving accurate and reliable quantification of DNA and RNA just got easier. QIAGEN provides the optimal solution for real-time PCR - from optimized kits and assays to fully automated PCR setup and outstanding real-time PCR analysis. Discover more at www.qiagen.com/automation.
Views: 27635 QIAGEN
Digital PCR vs. Real-time PCR - Ask TaqMan #30
 
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Ask your question at https://www.thermofisher.com/ask As a researcher, you’ve been given many choices in terms of tools and techniques. And for some who are intimately familiar with real-time PCR, you are probably hearing about digital PCR and its emerging applications. So the question is, when do you use one or the other or both. Real-time PCR – also called quantitative polymerase chain reaction or qPCR – is one of the most powerful and sensitive gene analysis techniques and is used for a broad range of applications. Digital PCR is the next generation of PCR technology involving absolute quantitation of nucleic acid target sequences. As digital PCR gains clout among researchers, many interested scientists ask “When should I choose digital PCR over real-time PCR?” The overwhelming advantage of real-time PCR is that it is a broadly accepted technology with well-established protocols and data analysis techniques. Other advantages include • a wide dynamic range for detection • a low per-sample experiment cost. • high sample throughputs The major advantages of digital PCR include: • no reliance on standard curves and reference samples; • high tolerance to biological and sample prep inhibitors; • and improved performance for applications requiring higher sensitivity and precision You can continue using real-time PCR for routine quantification applications, and add digital PCR for applications requiring enhanced performance. Absolute quantitation using real-time PCR requires standard curves and reference samples, but digital PCR allows you to quantify samples without using a standard curve. In this example using real-time PCR, notice how many wells are required to generate the standard curve. This assumes you have appropriate reference standards for your sample of interest, which may not always be the case. Each standard also must be of known quantity, such as absolute quantitation of mRNA copy number. Generating such standards is a lot of work and the standards may deteriorate over time, thus changing the amount of nucleic acid present in your standards. In the case of digital PCR, your sample is partitioned into thousands of separate reaction vessels. Taking an end point PCR read for presence or absence of reactions allows for the absolute measurement of template copies per microliter. Digital PCR is also highly tolerant to PCR inhibitors by virtue of the massive partitioning we just discussed. These data illustrate how digital PCR is robust in the face of increasing inhibitor concentrations; whereas, the performance with real-time PCR dramatically drops off at high inhibitor concentrations. Digital PCR can also provide improved performance in cancer research, where rare mutations often need to be detected in a background of wild-type DNA. Digital PCR has the sensitivity to detect extremely rare target sequences. . So, in the end digital PCR can enhance your quantitative PCR results, enabling applications that benefit from higher sensitivity, precision, and absolute quantification. If you’ve got more qPCR or digital PCR questions, remember, just ask TaqMan. Submit your question at https://www.thermofisher.com/ask and subscribe to our channel to see more videos like this.
Real-time PCR
 
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This video belongs to the section entitled "Molecular tests" that is part of the DVD "Avian Influenza sampling procedures and laboratory testing" funded by FAO and the Istituto Zooprofilattico delle Venezie (IZSVe). (c) FAO www.fao.org
Real time PCR
 
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Views: 62221 Emilija Miltenytė
Soya Analysis: Real-time PCR Amplification & Detection
 
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Real-time PCR for soya analysis: amplification and detection with SureFood® Allergen
Views: 873 R-Biopharm AG
How to analyze gene expression from cultured cells
 
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Learn how to prepare RNA for gene expression analysis from cultured cells in 7 minutes. The Cells-to-CT™ 1-Step TaqMan Kit is a simple, quick alternative to traditional RNA extraction. This video will show you how to get great qRT-PCR results in a fraction of the time. View the complete protocol here: https://tools.thermofisher.com/content/sfs/manuals/MAN0010650.pdf
1-step vs. 2-step RT-PCR—which one is right for your application?
 
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http://www.thermofisher.com/us/en/home/life-science/pcr/reverse-transcription/superscript-iv-one-step-rt-pcr-system.html?cid=BID_R01_PJT2521_999_VI_YUT_OD_KT_171120_TF_A_VD165 The one-step RT-PCR approach is faster, requires less pipetting and minimizes possible contamination. With the new Invitrogen™ SuperScript™ IV One-Step RT-PCR System you can get superior one-step RT-PCR results even with challenging RNA samples. Learn more at thermofisher.com/ssiv-onestep
How to achieve Absolute quantification in qPCR
 
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Learn about an alternative to the Ct method and save money by reducing reagent costs, number of replicates, and laborious standard curves. Get better information form your existing qPCR instrument and data and learn how to improve every qPCR reaction into an absolute PCR determination.
Views: 6800 DNA Software Inc.
qPCR technique animation tutorial
 
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Quantitative PCR (qPCR) animation tutorial - This animated lecture explains about the step by step process of quantitative realtime PCR or qPCR technique. Quantitative realtime PCR help us monitoring the amplification of target DNA in the PCR mix with time in realtime by generating fluorescence light which is detected by the fluorescent detector attached to the PCR machine. qPCR is the process of understanding the amplification of DNA content quantitatively in the PCR reaction mix. Here we explain the quantitative PCR reaction carried out with sybr green probe and Taqman probe. Animation source - www.sumanasinc.com Narrated by - Suman Bhattacharjee For more information, log on to- http://www.shomusbiology.com/ Get Shomu's Biology DVD set here- http://www.shomusbiology.com/dvd-store/ Download the study materials here- http://shomusbiology.com/bio-materials.html Remember Shomu’s Biology is created to spread the knowledge of life science and biology by sharing all this free biology lectures video and animation presented by Suman Bhattacharjee in YouTube. All these tutorials are brought to you for free. Please subscribe to our channel so that we can grow together. You can check for any of the following services from Shomu’s Biology- Buy Shomu’s Biology lecture DVD set- www.shomusbiology.com/dvd-store Shomu’s Biology assignment services – www.shomusbiology.com/assignment -help Join Online coaching for CSIR NET exam – www.shomusbiology.com/net-coaching We are social. Find us on different sites here- Our Website – www.shomusbiology.com Facebook page- https://www.facebook.com/ShomusBiology/ Twitter - https://twitter.com/shomusbiology SlideShare- www.slideshare.net/shomusbiology Google plus- https://plus.google.com/113648584982732129198 LinkedIn - https://www.linkedin.com/in/suman-bhattacharjee-2a051661 Youtube- https://www.youtube.com/user/TheFunsuman Thank you for watching qPCR technique animation tutorial
Views: 99901 Shomu's Biology
qRTPCR data analysis
 
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Views: 14785 profbiot
Hints for Real-Time PCR (qPCR) Assay Design -- Ask TaqMan®: Ep. 2
 
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Submit your Real-Time PCR questions and watch the rest of our videos at http://ow.ly/bQh0l. Get some helpful hints for Real-Time PCR assay design. It's a common question Life Technologies gets from people who've done a lot of gel-based PCR over the years and designed hundreds of primer sets, but never for real-time PCR. You might be asking yourself "Do I need to do anything special?" First off, size definitely matters when it comes to amplicons in real-time PCR. Be sure not to make your products too large, as this can contribute to a less-than-optimal PCR efficiency. Instead, try to keep them somewhere between 50 and 150 bases long. Second -- and this is critical for quantitative experiments -- don't design oligos to a mismatch or to a known polymorphic site, as this will cause inefficient binding during the first few PCR cycles and shift Cts to the right. Instead, always confirm that you have the correct sequence. Third -- be careful about designing to sequences that aren't unique. Be especially mindful of homologues, which often differ very little, if at all, in certain regions, and will therefore cause erroneous signal. Try to always design at least one oligo to a region that's unique enough so that your assay won't give unwanted signal. Finally, you never want to design oligos to low-complexity sequence, including repetitive regions that certainly won't be unique in your genome or transcriptome. There are lots of ways to get assays that fulfil all the criteria I mentioned. One way, of course, is to do all the bioinformatics work yourself on the context sequence, then use a program such as Primer Express Software to do the assay design. You then just order individual oligos. One of the really nice things about Primer Express Software is that it designs every set of oligos to work at Universal Cycling Conditions, meaning you never have to optimize real-time PCR conditions. Speaking of convenience, you should know about 2 other handy options for getting high-quality TaqMan® Assays: by either having Life Technologies design them for you through one of their Custom services, or by purchasing a pre-designed Assay. In either case, you can spare yourself the trouble of doing bioinformatics, your assays will all work at Universal Conditions, and they'll amplify with 100% efficiency— all of which should make you very happy. If you want to try out a pre-developed TaqMan® Assay for gene expression, we have excellent coverage for lots and lots of species. We even have pre-developed TaqMan® Assays for other applications, including SNP genotyping, microRNA and other small RNAs, copy number assays, and lots more. And don't worry -- if we don't have your assay already, we can always custom design it for you.
CFX Manager™ Software Part 4: Doing Data Analysis
 
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For more information, visit http://www.bio-rad.com/yt/4/TechSupport-CFX-Mgr This brief tutorial walks through the various data analysis options in CFX Manager 3.1. Bio-Rad’s CFX Manager™ Software provides intuitive qPCR setup and rich data visualization tools to reduce confusion and anxiety while performing real-time PCR experiments. CFX Manager Software is included with al Bio-Rad CFX Real-Time PCR Detection Systems: • CFX96 Touch™ System • CFX96 Touch™ Deep Well System • CFX384 Touch™ System • CFX Connect™ System Features and Benefits: • One-click experimental setup and data analysis with the Startup Wizard • Easily customized data analysis and export preferences • Application-specific data analysis for gene expression and SNP genotyping studies • Graphical data representation helps you quickly interpret and understand your data http://www.bio-rad.com/en-us/category/pcr-analysis-software?WT.mc_id=sm-GXD-WW-cfx-mgr-vyt_20160121-Hui6dXAabnA We Are Bio-Rad. Our mission: To provide useful, high-quality products and services that advance scientific discovery and improve healthcare. At Bio-Rad, we are united behind this effort. These two objectives are the driving force behind every decision we make, from developing innovative ideas to building global solutions that help solve our customers' greatest challenges. Connect with Bio-Rad Online: Website: http://www.bio-rad.com/ LinkedIn: https://www.linkedin.com/company/1613226/ Facebook: https://www.facebook.com/biorad/ Twitter: https://twitter.com/BioRadLifeSci Instagram: @BioRadLabs Snapchat: @BioRadLabs
Views: 25126 Bio-Rad Laboratories
Real Time qPCR  optimization, Calculating PCR Efficiency
 
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http://technologyinscience.blogspot.com/2012/12/generating-standard-curve-to-analyse.html Generating Standard Curve to analyse the reaction optimization - Real Time qPCR, Calculating PCR Efficiency
Views: 32853 Bio-Resource
Absolute Quantification of mRNAs - Ask TaqMan #26
 
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Submit your question: http://bit.ly/1cgFftk Relative quantitation is the most common application with real-time PCR, but sometimes fold change data is just not enough. For instance, let's say I'm looking at samples infected with HIV and I need to know exactly how many copies of virus are present in the sample. What other options are there, when you need more concrete answers? That brings us to this great question from Jamsai Duangporn at Monash University who asks "Can TaqMan assays to be used to determine the absolute quantity of the mRNAs?" TaqMan assays can be used for a technique called Absolute Quantitation, sometimes also known as Standard Curve analysis. ABSOLUTE QUANTIFICATION involves the precise molecular measure of a target concentration. In an ABSOLUTE QUANTIFICATION experiment, samples of a known quantity are serially diluted and then amplified to generate a standard curve. The unknown samples can then be extrapolated into quantities based on the slope of this curve. The main hurdle in an ABSOLUTE QUANTIFICATION experiment is the generation of this standard curve. Although it seems simple in principle, there are a lot of things to consider! Your standard needs to meet the following criteria: First, the quantity of a sample must be known by some independent means. For this step, the concentration can be measured by with a spectrophotometer and converted to number of copies using the molecular weight of the DNA or RNA. You can also refer to our handy guide called "Creating Standard Curves" for more details on how to do this. Second, the standard should closely resemble the target from a biological standpoint, and it is very important that the DNA or RNA be a single, pure species. For example, when measuring gene expression of RNA transcripts, you would want to use in vitro transcribed RNA. Take care here because purity will be an important factor in the accuracy of your measurement. Third and finally, don't forget that your excellent pipetting skills can be put to good use here! One of the major pitfalls for scientists setting up standard curves is that they do not pay enough attention accuracy of pipetting of technical replicates. For the best results from your standard curve, ensure that your pipets have been recently calibrated. Be very careful when making dilutions and pipetting into the plate, and ideally make use of low retention tips. Now that we have our standards, let's setup a dilution curve in our plate. We recommend to run in triplicate, with 10 fold dilutions and at least 5 points. When set up correctly, all ABI Real-Time PCR instrument software will generate a standard curve for you from these points. The equation of the linear regression line through those points is then used to automatically calculate the quantities of any unknowns on the plate, in the same units. For example, in this curve I have 5 points, starting with 20,000 copies of my target as the highest concentration going down to 1250 copies. The standard curve plot is showing the input on the x-axis as [log X] and the Ct values on the y-axis. Quantities are then determined from this equation: We'll remove any outlying replicates or points when necessary. Solving for X using the Ct values of our unknown samples will give us the missing quantities. Notice that they will be in the same units as our standards; copies for copies, ng for ng, and so on. So now we have determined the absolute quantities of our unknown samples!
Amplify Sample with The StepOnePlus™ Real Time System  (qPCR step 6)
 
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The StepOnePlus™ Real-Time PCR System is a 96-well Real-Time PCR instrument perfect for both first-time and experienced users. The StepOnePlus™ Real-Time PCR System can be setup in a variety of configurations and comes ready to use, out of the box, with intuitive data analysis and instrument control software. Utilizing robust LED based 4-color optical recording, the StepOnePlus™ Real-Time PCR System is designed to deliver precise, quantitative Real-Time PCR results for a variety of genomic research applications. The other videos in this series are: Introduction to the steps of the qPCR workflow (qPCR step 1): http://youtu.be/UOhD0jwCtEg Isolate RNA with the PureLink™ RNA Mini Kit (qPCR step 2): http://youtu.be/I174cAKluoo Quantitate RNA with the Qubit® 2.0 Fluorometer (qPCR step 3): http://youtu.be/bSSlO2fqEN8 Superscript Vilo cDNA synthesis kit (qPCR step 4): http://youtu.be/GUvkYFUBFLw Fast SYBR Green vs. TaqMan® Fast Advanced Master Mix (qPCR step 5): http://youtu.be/DsKDWVLMvuc Preparing the card for the Viia™ 7 with TaqMan® Assays (qPCR step 7): http://youtu.be/Vg1AJQ1CXjo Amplify sample with the Viia™ 7 Real-Time PCR system (qPCR step 8): http://youtu.be/MSHEhqDNeak -------------- Transcript: Alright. Let's recap a typical real-time PCR or qPCR experiment in a plate. What you will require is the actual plate, the plastics and the plastic holder. The optical adhesive cover and the optical adhesive sealer. In addition for the reagents, if you're doing a Taqman experiment, you will require the Taqman fast advance master mix. TaqMan gene expression assay. Sybr experiments will require the sybr ring master mix, and, 2 primers, which I have designated, primer 1 and primer 2. P 1 and P 2. Also. When we fire your CDNA sample and finally some water. These pipe [indiscernible] are to show you that you essentially pipette the reagents into the plate. You then seal the plate. This is the final product, which we then run on the instrument and in this case the Step One Plus. And now I'm walking over to the Step One Plus. I'm going to load my plate in the instrument simply by pulling open the door. And setting the plate, The A goes up in the top as usual. And closing the door. Now I'm just going to use the touch screen function of the Step One Plus. And I already have TaqMan Fast CDNA protocol right here on the main menu. So, I am just going to choose that one and press save. OK I saved my experiment. I start the run now. Say okay. Now what's happening is, the cover is heating. And as soon as the cover has come up to the proper temperature, you will see that this wide band becomes smaller as the plate is moving up. And as it says here on the screen, the required run temperature of the heated lid is 105. We're right now at 90.5. So after a few more degrees, the run will begin. So now that the run is completed on the step 1 plus, I'm going to collect results by putting in my USB drive, and hitting the collect results button. The system is busy, it's writing the USB. Please don't remove the USB device. And you can see as the data is being written, we have the little bars coming across the screen. Now my experiment is ready. I may remove my USB storage. I hit okay. My data is on here. I'm going to go take it to my computer at my desk and analyze my results. For more information, visit: http://www.invitrogen.com/site/us/en/home/Products-and-Services/Applications/PCR/real-time-pcr/real-time-pcr-instruments.html
qPCR Training Video
 
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The Southern California Coastal Water Research Project coordinated two demonstration projects in 2010 and 2011 using a rapid method (quantitative polymerase chain reaction) to assess beach water quality at sites in Orange and Los Angeles Counties. This video was produced to train laboratory staff to execute the method.
Views: 202080 sccwrp