Error Bars Ct Values
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a bar graph? I have my raw data according to two groups of different types of animals that are being tested with pharmacological compounds or PBS. I am using the 2deltaCT method. I've seen standard error values different papers with many ways of graphing results, lets say for instance: relative expression percent error value vs gene of interest, fold change vs gene of interest, RQ vs Gene. Does anyone have an opinion on the best way
Value Of Standard Error Formula
to depict your relative quantification data? Topics Molecular Biological Techniques × 7,033 Questions 33,963 Followers Follow PCR × 4,995 Questions 71,931 Followers Follow Methods × 3,956 Questions 132,103 Followers Follow Gene Expression × 1,716 Questions 25,297
Value Of Standard Error Calculator
Followers Follow Real-Time PCR × 2,144 Questions 3,370 Followers Follow Feb 27, 2014 Share Facebook Twitter LinkedIn Google+ 0 / 1 Popular Answers Jochen Wilhelm · Justus-Liebig-Universität Gießen Short answer: Corectly applied, barcharts only allow you to show ddCt values. Longer: If you want to relate expressions among several groups, it's best to show dCt. If the comparisons at all done to the same reference, best show ddCt. Never ever use simple qpcr error bars barcharts (as used so terribly often in biomedical papers). If you have small sample sizes, you can show dCt values simply in 1D scatterplots. For larger sample sizes, dCt values can be shown in boxplots, or in dot-plots with error bars (indicating median and IQR or the mean and 95%CI). In case of ddCt values the only option are dot-plots with mean and CI (since there are no "individual measurements" and you can only provide the mean ddCt). If you are forced to show 2^dCt or 2^ddCt, then I'd suggest to calculate all statistics (like medians, IQRs, means, CIs) for the dCt (or ddCt) values and potentiate these results to show them in a plot. For the means and CIs this gives you the "geometric means" with according CI (what is not symmetric around the mean). If you are forced to use barcharts, you cannot show dCt because this quantity has no interpretable zero value (so a hight of a bar, even the direction [positive or negative] provides no interpretable information; so the purpose of the barchart [=showing bar areas] is completely off-topic). You can neither 2^dCt or 2^ddCt on a barchart, too, because here (1) not the zero but the on is the reference value, and even if you would place the x-axis at
group with error bars on the relative expression histograms in a qPCR study. I have a question regarding to the representation of the relative expression histograms. We did a comparative qPCR study, and, using the Pfaffl's efficiency corrected Ct
Fold Change Error Bars
formula, I calculated the relative expression values for all of my samples. Since I standard deviation of fold change have biological replicates, I get error bars for the treatment groups on the relative expression values, but in many papers I noticed gauß' error propagation that they also use error bars for the control groups (value 1 with an error bar). How? Topics PCR × 4,995 Questions 71,931 Followers Follow Real-Time PCR × 2,144 Questions 3,370 Followers Follow Feb 12, 2013 Share https://www.researchgate.net/post/How_do_I_publish_qPCR_data_in_a_bar_graph Facebook Twitter LinkedIn Google+ 2 / 0 All Answers (3) Jo Vandesompele · Ghent University Biogazelle's qbasePLUS software (http://www.qbaseplus.com) can do this. If you want to do this manually in a spreadsheet, I would need a bit more information on how EXACTLY you did you calculations. If you calculated relative quantities for all you samples at once (e.g. according to Hellemans et al., Genome Biology, 2007), you will also have variable results for your https://www.researchgate.net/post/How_to_represent_control_group_with_error_bars_on_the_relative_expression_histograms_in_a_qPCR_study control group and hence an error bar for this group. Feb 13, 2013 Jack M Gallup · Iowa State University Dr. V, your software is the best in the world for this. It's good to let the cat out of the bag at this point. And it is always good to hear your opinion on qPCR stats. The error bars for the controls is always propotional to their error bars before the control was divided by itself. I believe this is also equal to the coefficient of variance. E.g., if the error bar for a control value of 0.5 was +/- 0.2 (before control was divided by itself), then, when the control becomes "1" by self-division, the error bar becomes (by proportion or coefficient of variance rules) +/- 0.4. But if the error bars are the result of transformation from log to linear scale, the error bar above and below the median is not symmetrical... technically, and thus a more lengthy explanation is needed. Error just doesn't simply disappear... must always be accounted for - and very tricky, unless you use Dr. V's and Dr. H's software. Feb 14, 2013 Jochen Wilhelm · Justus-Liebig-Universität Gießen I do not see the point of showing the "normalized controls" as a bar of height 1 - with or without error bars.
)Visit this topic in live forum Printer Friendly VersionHi, I need help with some qRT-PCR error bar calculations. I have carried out all http://www.protocol-online.org/biology-forums-2/posts/33479.html of my qPCR with my gene of interest and of my housekeeping http://biology.kenyon.edu/HHMI/Real_Time_PCR/Sample_Data.htm gene and have calculated my fold-changes for my gene of interest with the delta-delta-Ct calculation. However putting error bars on my graph showing the fold-changes is confusing me. What data should I be using to calculate this? I can't use the normalized Ct values as this is error bars not correct for fold-changes and when I convert my Ct values (for my calibrator and experimental) using the delta-delta-Ct calculation and then get the SE for that my error bars are bigger than my fold-change. Can anybody shed some light on this and where I am going wrong? I can't use the software that comes with the qPCR machine as value of standard it is getting confused with how my plates are arranged. Thank you in advance!
-toffee89- Just curious, what software comes with your machine (i.e. what is your machine) that gets confused by plate arrangement? -Trof- Hi, have you read Livak & Schmittgen 2001? Changing the exponential process (PCR amplification) into linear comparison (fold change),as it happens in the method (2^-delta-delta-Ct),need to be also applied for the upper and lower values of the error. You need to calculate the standard deviation of your delta-Ct values and calculate the positive and negative error values from delta-delta-Ct +SD anddelta-delta-Ct -SD using the same2^ conversion. If this is not done, especially in the case when the "treated" group is lower than 1 (control) simple SD error bars may cross the X-axis. The correct error bars for this type of data are always different size in + and - direction. Hope this helps! -miRman- Visit this topic in BioForum Printer Friendly Version About Terms of Service Privacy Feedback Sponsorship © 1999-2013 Protocol Online, All rights reserved.required to reach a given level of fluorescence (Y-axis) within the log-linear range of amplification? More abundant mRNAs require fewer cycles of PCR amplification to generate the same amount of product, and therefore, fluorescence. Since each cycle represents a doubling of the PCR product, a one cycle difference in Ct represents a two-fold change in expression. Fig. 1. Cytochrome P4501A6 mRNA is induced by dioxin. Total RNA from was isolated from control and dioxin-treated Xenopus laevis tadpoles and reverse transcribed using random hexamer primers. cDNA products from 10 ng of total RNA were used as a template in real-time PCR with SYBR Green DNA detection dye. Each PCR reaction was performed in triplicate. Note the leftward shift in the CYP1A6 amplification curve in the dioxin-treated samples. Average Ct values for CYP1A6: Unexposed samples: 31.420 Dioxin-exposed samples: 27.240 Fig. 2. Ct values for CYP1A6 are normalized to beta-actin by subtracting the average Ct value for each treatment. Expression of this housekeeping gene should not change in response to dioxin exposure. Small variations in the amplification curves and Ct values (compare panels A and B) are used in normalizing the Ct values for CYP1A6 (derived in Fig. 1). Average Ct values for beta-actin: Unexposed samples: 22.268 Dioxin-exposed samples: 21.565 This is accomplished by subtracting the mean Ct for beta-actin from the mean Ct for CYP1A6. Resulting values for each treatment group are called the delta Ct values. delta Ct values: Unexposed samples: 31.420 - 22.268 = 9.152 +/- 0.208 (SE) Dioxin-exposed samples: 27.240 - 21.565 = 5.675 +/- 0.211 (SE) (Panel A) (Panel B) Next, the relative CYP1A6 expression in control and dioxin-treated tadpoles is quantified using the delta-delta Ct method. First, the delta Ct value for each treatment group is normalized to that of a single group through subtraction. In our experiment, we will normalize CYP1A6 expression to the untreated samples: delta-delta Ct valu