# Real Time Pcr Error Propagation

mean of **Ref.-Genes, sub err would** be calculated using: (squroot((Error(a)^2)+(Error(b)^2)))/2, right? Ct values are on a log scale so a subtraction is actually already equivalent to dividing the unlogged values.TonyDeleteReplyrashmi27 January 2014 at 07:28Hi TonyThanks for all the information. If I can't use these as error bars, what would you recommend plotting as a graph, since graphs now-days all need error bars? permalinkembedsavegive gold[–]Faytezsm 0 points1 point2 points 2 years ago(0 children)I've only ever seen the error calculated from the fold change values. weblink

I don't know about later versions of REST. If you don't have a solid hypothesis about what you are looking for, but instead compare it against everything you can, then I'd expect you to find a high number of I have two conditions (time=0, time=1), 1 GOI, 2 reference genes with a geo average of REF.In time=0: GOI = 20, REF = 15In time=1: GOI = 19, REF = 15As How do this analysis work if I have two groups of patients (healthy and diseased, say 10 patients in each group)?ReplyDeleteRepliesTony McBryan27 January 2014 at 10:36This is the situation I've described

Replicates & Error Propagation One place that a number of mistakes seem to creep in is the treatment of replicates within PCR experiments. Assuming several replicates, on let's say the PCR and RT level, have been measured, the sub err, after averaging these one after another would be calculated by basically using the same Because when I do that i get 0.81 for normal conditions and 0.88 for stress which is what i would expect.DeleteTony McBryan22 January 2014 at 11:06I would argue that your experiment In your spreadsheet they are listed as 0.015.

The key fact to remember is **to always use** the arithmetic mean on anything on a linear scale and always to use the geometric mean on anything on an exponential scale. So let xi (be the group indicator for the i-th sample and yi the corresponding dct. Which means I have my Ct result of target gene and HKGs obtained from qPCR experiment. References Livak, Kenneth J., and Thomas D.

In your spreadsheet you calculate the SEM for condition A and condition B and then pool these t current community chat Biology Biology Meta your communities Sign up or log in I took a look at the reference you gave but it wasn't clear given that they don't really discuss the delta-delta Ct method. How do I get the p-value on the ONE delta delta CT value if it was calculated based on a average of bunch of CT values (I took the average of If something is not readable, please let me know!

If you have different concentrations then your control gene should be different which corrects the concentration problem.It would be wrong to divide the Ct values. So sorry if I have caused any inconvenience. Alphabet Diamond Subdividing list with another list as a reference What does "Game of the Year" actually mean? Another, more accurate option, is to subtract the error (after calculating it for the delta delta values) from your delta delta Ct, take 2that answer .

I have never worked with Taylor series before, and as I am missing their background logic I find them confusing. I just incorporated the sub err of ref genes a,b into the equation you mentioned in you upper text (replicates and error propagation)!? Technical questions like the one you've just found usually get answered within 48 hours on ResearchGate. You won't be able to vote or comment. 123Error Bars for q-pcr/rt-pcr? (self.labrats)submitted 2 years ago by baller_unicornI am a new graduate student and I am running a lot of rt-pcr/q-pcr but I have pretty

Similarly there are open source solutions such as pyQPCR. have a peek at these guys permalinkembedsaveparentgive gold[–]natched 1 point2 points3 points 2 years ago(0 children)How you calculate error bars is kind of determined by how you are presenting the data. A new mathematical model for relative quantification in real-time RT-PCR. Need help with your experiments?

I say 33%, my teacher said 12.5%. I am trying to analyze using the comparative ct method but I am not sure from which data I should calculate the error bars. i.e. check over here Do I load the same amount (ng) of cDNA into each qPCR reaction?

Do the calculations the exact same way you would normally, except add the + and - error in the Ct value to the mean Ct, and carry it through the rest So the ratio of target gene to reference gene in each sample is therefore $$$2^{CT(target)} / 2^{CT(ref)}$$$. However, one thing is still not clear to me:If I am right, the spread of the final concentration values is made up of two components: (a) the "biological" variability already present

## I have 8 or so different experimental primer pairs and I have actin as a control.

For your information, I am quantifying using absolute quantification, 3 housekeeping genes(HKGs) were tested together in this experiment. Taylor series expansion of error propagation calculations for qPCR are complex; still a topic very advanced statisticians are wrestling with; Dr. I have to point out several things: The fraction of incorrect DNA molecules does not depend on the number of cycles (as long as the number of cycles is higher than However, we will then usually also include an Input sample which the ChIP sample is compared with.

We then calculate the ratio between our two sample by calculating the quotient between the ratio of target gene to reference gene between the two samples as such: $$$$Ratio = {2^{CT(target,untreated) Then find the geometric mean (which is then used as the reference gene CT as per Vandesompele et al. qBase relative quantification framework and software for management and automated analysis of real-time quantitative PCR data. http://lebloggeek.com/real-time/real-time-clock-error-lenovo.html Genome biology 3.7 (2002).

The exact order that you do the subtraction in actually doesn't make a huge difference and you'll probably see other people do it slightly differently. I am currently processing some qRT-PCR data and I have exported the Ct values and have replicates for all my samples. permalinkembedsavegive goldaboutblogaboutsource codeadvertisejobshelpsite rulesFAQwikireddiquettetransparencycontact usapps & toolsReddit for iPhoneReddit for Androidmobile websitebuttons<3reddit goldredditgiftsUse of this site constitutes acceptance of our User Agreement and Privacy Policy (updated). © 2016 reddit inc. REST 2009 et al.).

thank you Jun 16, 2016 Can you help by adding an answer? Technical questions like the one you've just found usually get answered within 48 hours on ResearchGate. Delta Delta CT The Livak method is more commonly known as the "Delta Delta CT" (ΔΔCT).