How to calculate IC50
ฝัง
- เผยแพร่เมื่อ 28 ก.ย. 2024
- In this tutorial I demonstrate how to plot an IC50 curve for drug assay data in Graphpad prism 6.
I will like to point out an error i made in the video (skip to 8:35) where i assigned a microMolar unit to the LogIC50 value. Honestly, LogIC50 values have no unit so please note it. I apologize for this.
very well explained. could you also please explain EC50 calculation using prism.
Hi. EC50 calculation is very Similar. After normalizing your data, do Analyse -> NonLinear Regression -> Dose-response Stimulation -> Log(agonist) vs Normalized response.
@@Dr.DanielAddoGyan do we choose Log(agonist) vs Normalized response - Variable slope, for finding EC50 or Log(agonist) vs Normalized response ?
tin_foil _hat with variable slope
@@Dr.DanielAddoGyan thank you very much!
@@Dr.DanielAddoGyan could you explain CC50 calculation using prism, please.. i dont know how to proceed after the "NonLinear Regression" step.
Daniel, thank you for the tutorial. It was very useful and it was very well explained. Thanks a lot
Hello, i am glad you like it
Very simplified. Good work
Thanks Gina
Thank you so much.. well explained
Thanks for your video very helpful. I have a question. I have a drug concentration 1uM, how do I to calculate this one. For example, in the video you change 0 to 1. Indeed I have 1 in my plate. Hope to hear from you.😊
Thanks for watching my video. I admit that, even though changing 0 to 1 works for some datasets, it does not work for others. A better approach for your case will be to add a small number (0.0001 for example) to all your y-values, run the analysis for IC50 and then subtract that number from the IC50.
Where can I find the SEM value for the IC 50 value?
On the nonlinear fit result sheet you can find the std error of the logIC50
i have uploaded a video explaining how to find the SE and SD of IC50: th-cam.com/video/H999TYf7IWQ/w-d-xo.html
Thank you so much, this was really helpful
Glad it was helpful!
This is excellent Daniel. Thanks a lot
Thank you Daniel!!!
Well explained. I have a question regarding my work....why IC50 sometimes appears not corresponding to 50 as it should be in the curve? I got IC50= 7 while the curve shows that more than 30 mg/ml is the IC50 concentration.
Thanks for the question. Did you input raw absorbance values or were the values already transformed (for example to percentage inhibition), did you normalize the data or not? could you give some more details about what you did?
@@Dr.DanielAddoGyan I am having the same problem and I input already transformed values (percentage). Do you think that might be the problem?
Hi there, thank you for the video. Can I use the same steps to calculate lethal temperature 50 (LT50)? Thank you.
Yes, you can
Thank you! Can you tell me how to calculate CC50 using this guide?
Thanks for the question. You can calculate CC50 with the same method as IC50. The difference between IC50 and CC50 is how you interpret the results
Thank you for the tutorial... How do I convert absorbance readings to be like yours?
I am glad the video was helpful. Your absorbance doesn't have to be exactly like in the video. whatever values you have should work as long as you are using the corresponding concentration values. Also there is nothing wrong with your graph if it falls on the negative side of the graph, the software can still calculate the IC50. If you want to transform the absorbance, you could divide or multiply them by some common factor. Make sure you apply the same transformation to every absorbance value. hope this helps
thank you for your explanation. kindly tell how to calculate SD value from draph alog with ic50
Will upload soon
i have uploaded a video explaining how to find the SE and SD of IC50: th-cam.com/video/H999TYf7IWQ/w-d-xo.html
Is it possible to display the IC50 as a dotted line on the graph itself?
yes it possible to do that
Thanks a lot. It is very useful.
Thanks
What if I have a concentration of 1? it will be changed to 0
Hello, It is better to add a small value (0.001 for example) to each value of x before log transformation.
So helpful, Thanks a lot !!!
Welcome
Thanks. 👏👏👏👏👏👏
You're welcome
I'm still having issues with getting a good R2 value and neat curve. My log transform data is also negative but the values of my concentrations aren't less than 1.00. If I wanted to express my concentrations in another unit in graph pad how could you do so ?
Hi Rose. Usually a bad R2 is because your data does not very much follow a sigmoid pattern. About the concentrations, what unit are yours in?
I dosed my concentrations in um but i'm not sure if the software is set to a specific unit
Thank you!!!
I want my x axis to have the concentration numbers instead of the log
Use [inhibitor] vs Normalized response or Use [inhibitor] vs response
If you do log transformation for zero, it gives you Infinity... Thats' why, it was displayed as empty
i agree. thanks
Hi Dr. Thanks for the explanation. At the early stages, you normalize the transform data to make it in a form of percentage. As for my data, it is already in a form of percentage. May I know if I still need to normalize the transformed data?
Hello. You can skip normalization of the y-axis values if it is already in the percentage.
After hours of searching for a clear explanation this is just what I needed thank you
I am Glad i could help
Why the ic50 value at the end read 7.8 whereas as the reads of absorbanse make to predect it nearly 25000 because it give inhibition to nearly halve of the high read 66585
Hello thanks for the question. However i am not sure i clearly understand the question. Could you please rephrase the question?
Thank you for the video, it was very helpful.
Changing the value to 1 is not an appropriate solution to ridding yourself of the missing value. If anything you should do a different transformation like log(x+1) or log(x+.01). The first of these 2 would be the equivalent of adding 1 to each of your X values and then taking the log transformation. 1 is also relatively large in comparison with 0, so you should at least choose something closer to 0 like 0.001 (and this is still improper). And yes, you might get similar results, but it is the better practice.
Thats a nice suggestion. Thanks
Thank you for the tutorial! If I don't have a dose-response curve, can I still calculate?
Could you explain further? Do you mean the curve doesn’t pass through the points?
Hello!
GraphPad Prism software only reports standard error for LogIC50 and not the IC50 itself. Is there a way to report the standard deviation (or error) using prism?
Hello!. yes there is a way. Please checkout this link if you haven't yet: www.graphpad.com/support/faqid/34/
Thank you for the tutorial. Why did you transform the data? Pls explain
Using a logarithmic x-axis (concentration) spreads out the data so the shape of the curve and the quality of the fit are readily visible when the concentrations cover a wide range.
Hello sir, I would like to calculate EC50 of my data but my concentration is 0.000, 0.001, 0.010, 0.10, 1,10
in which format should I put my data I can analyze EC 50
Hello. you can still use these values as they are. the curve will sit in the negative left side of the graph which is fine. but if you like, you could convert the values by multiplying by a factor for eg. 1000. this would give you the values 0,1,10,100,1000, 10000. after you get the IC50, divide by 1000. Alternatively just convert your units to a lower one. eg. 0.1 milliliters would convert to 100 microliters, then the IC50 would be in microliters. hope this helps
thank you for the very clear and informative video. May I ask - I have seen some videos using the MEAN of the 3 replicates to set the 0% and 100% values, rather than using the lowest and highest values respectively. When do we use which? Thank you.
Nice but, what happen if you have 1uM of dose, how is the procedure
I am glad you liked it. The procedure is the same
Are the y values in percent viability or just absorbance
Thanks for watching my video. The Y-axis represents the absorbance
Hello ! Thanks for the tutorial ! I have a question ! In the first column, i put my concentration of inhibitory (decreasing between 5µM --> 0.05µM), but, when i ask to transform in log, i obtain negativ results for all concentration behind 1µM. I think its because log of >1 doesnt exist. Do you have a solution for this issue ?
Of course, i cant obtain an good EC50 curve if my results are negativ.
Thanks for your answer ! Sorry for my english.
I have the same problem too! Hoping for an answer :)
@@melissa2872 no answer After 1 year :( i you want i can watch on my curve monday how ive perform to do it !
Hey I had the same problem so instead I converted the concentrations to nM by *1000, this was u r n the positive side. Hope this helps!
Samuel I am very sorry for not answering your question till now. Some ways to solve this is to convert your concentrations to another unit by multiplying all your concentrations by a factor such as 1000. For example you can convert from M to mM by multiplying your concentrations by 1000. This will convert to values above 1.0. On the other hand, having log concentrations in the negative side does not give error in your IC50 or EC50 estimations. You can can also shift the enter graph to the positive side for example by changing where the x and y axis intersect. I have demonstrated how to do this in my videos on MIC and NIC estimation.
Thanks for your video very helpful. I have a question. I have a drug concentration 1uM, how do I to calculate this one. For example, in the video you change 0 to 1. Indeed I have 1 in my plate. Hope to hear from you.😊 😊
Thanks for watching my video. I admit that, even though changing 0 to 1 works for some datasets, it does not work for others. A better approach for your case will be to add a small number (0.0001 for example) to all your y-values, run the analysis for IC50 and then subtract that number from the IC50.
To find ic50 value, do we have to normalise the data, if yes means can u please say why..?? Or we can just transform the data and directly go for nonlinear regression (curve fit) without opting for normalise..??
My doubt is that, e
We will get ic50 value for both methods, I mean select the data, then going for analyze, then transform and directly go to nonlinear regression.. We will get ic50 value (eg: 350.2)..
Then the other method which is shown in the video like select data, analyze, transform, then normalise and then go for nonlinear regression curve. And we got ic50 value (eg. 290).. R² value is 0.989 something for both ic50 value.. so which method is correct???
Thank you
You should normalize when you are sure the highest value is the 100% response and the lowest value is the 0% response. Otherwise Fit the curve without normalization
Thanks for your video very helpful. I have a question. I have a drug concentration 1uM, how do I to calculate this one. For example, in the video you change 0 to 1. Indeed I have 1 in my plate. Hope to hear from you.😊
Thanks for watching my video. I admit that, even though changing 0 to 1 works for some datasets, it does not work for others. A better approach for your case will be to add a small number (0.0001 for example) to all your y-values, run the analysis for IC50 and then subtract that number from the IC50.
Thank you very much. Best explanation
You are welcome!
Hi Daniel..I would like to ask how can we see the SD from IC50 calculation?
Hi. Graphpad gives the std error of the logIC50
@@Dr.DanielAddoGyan Hi Daniel thank you. Yes prism gives the error for logIC50 but how can I have the one for IC50?
i have uploaded a video explaining how to find the SE and SD of IC50: th-cam.com/video/H999TYf7IWQ/w-d-xo.html
i have uploaded a video explaining how to find the SE and SD of IC50: th-cam.com/video/H999TYf7IWQ/w-d-xo.html
i have uploaded a video explaining how to find the SE and SD of IC50: th-cam.com/video/H999TYf7IWQ/w-d-xo.html
Is this applicable for MTT Assay as well??
Hello thanks for the question. Yes it is applicable to MTT assay
Hi, Thank you very much for your video. Do I need to transform my concentrations in mg/L to Molar to plot the EC50?
Thank you for your question. You can use any units you wish for concentration.
Revelation! Thank you:)
Is normalization a good choice here? Normalization chooses the highest point to be 100%, which is okay. But if your treatment does not inhibit the cell growth completely (some cells still alive), then it is wrong to assume the lowest point as 0%. Therefore, you could have wrong EC50 values.
I agree with you. In the video I did normalize because the compound inhibits completely. I should have explained in the video. Sorry about that and thanks for pointing it out
Excellent explanation. Wish I had found this at the start of my PhD rather than 2 months before the end!
Thanks. I am glad it helped
Is it necessary to normalize the data? What is the difference if I continue to analyze without normalizing the data? Thanks in advance!
You should normalize when your values account for 0% through to 100% response. If this is not the case, there is no need to normalize
Hello.. Is the ic50 value that I getting is reality or imaginary, I mean can I take it directly to used for example gene expression experiments or it should have some deals
Hello. Thanks for your question. However i am not sure i fully understand the question. Could you please rephrase the question
Thank you so much for your videos..i need a help regarding curve fitting..in my experiment i am checking how pH affects enzyme activity..it is poor at very acidic pH say,3, is maximum around 5-5.5, then again gradually wanes down..since prism automatically connects all the data points, i am getting a hump in the curve generated as there is an outlier..how do i keep my data point, but ensure that the curve doesn't paas through it to give a clean shape..
Let me check and get back to you
i have a problem, i am new to graphpad and after i click 'normalize', there is another option "subcolumns" before the "How is 0% defined?" how will I deal with this option? thank youuuu
Please which version of Graphpad prism are you using?
Thank you very much, it’s very useful information, iam using this software at first time so I have some doubt, I need to find out the IC50 value for my studies, my data have different concentration of nanoparticles (25,50,75,100,125 and 150 µg/ml) against Artemia mortality. After creating the IC50 value in GraphPad it shows Log IC50=0.2247, IC50=1.678, and R2=0.9440. At the same I make IC 50 value using scatter graph with a linear trend line in XL sheet. After, using the equation (Y=8.4762x-3.7778), I got the IC 50 value at 50.44 µg/ml. I don’t know which one is correct, please give me your suggestion.
Hello. From the R2 value (R2=0.9440) it is evident that your data follows a non-linear pattern on the XY graph, since it is close to 1. Take a look at the R2 value from excel. If it closer to 1 than 0.9440, then pick the value from excel , otherwise use the value from Graphpad prism. In this case using a linear trend-line for data that is not linear will lead to error.
I wish I found this months ago !!!
I am glad it helped
when I normalize my control is 100% and my first treatment indicates 48% but if I calculate the percentage by excel it indicates that my first treatment is 80%.
Can you please explain how you calculated it Excel?
Very well explained. Please also explain the calculation of MIC90 and MIC value. Thank you!
Will do soon
Hi Daniel, I used your video method for a university coursework, but got a really bad grade saying that I should not log values when calculating IC50, and even if I do, log value should have nM units next to it. Can you confirm that it should have no units, as I cannot find what is the right way to do it online..
Did you find the answer? please share
Sorry for very late response. log transformation of X might not be necessary in some cases depending on the nature of the Data and the requirements of the problem. Not sure why you got a bad grade but it will help if you share the question for your course work.
In 5.03 min, there is showing IC50. Is it indicating the concentrations of drug or absorbance?
IC50 indicates the concentration of the drug
Please give us reference paper
Well noted. will add references to subsequent videos
amazing video,
my OD values are around 0.1 and 0.3, can I know how to convert them to cells number as your data in the video?
I am glad you like the video. Actually you don't need to convert the OD values to be able to calculate the IC50
Please like, Share and subscribe so you don't miss out on future videos
hi. when i reach the step of selecting non linear regression and then a page come with selection of equation.. their is no equation coming, what could be the reason?
Which version of Graphpad prism are you using? It would be nice if i could see a snapshot of what you are talking about
WHY DID YOU CHOOSE THE LOG INHIBITOR VS NORMALISED RESPONSE- VARIABLE SLOPE? CAN YOU EXPLAIN
I chose "Log inhibitor" because, the x-values were log transformed and "Normalized response" because the y-values were normalized. "Variable slope" because in some cases it fits the sigmoid curve to the data better and gives a better r-square value. Variable slope assigns a dynamic hill-slope value rather than the usual fixed value of -1.
@@Dr.DanielAddoGyan ok I get it.
Hello Daniel, very useful video! Many thanks! I got R square value to be quite low (0.52), is there any way I can perform better fitting to get more reliable result?
So the R square of 0.52 means that the sigmoid curve model only explains about 52% of your data points. You can try other models such as polynomial, geometric, etc., to see which gives you the best R square
@@Dr.DanielAddoGyan thanks alot Daniel! Your comments and video are very helpful, I'm sure many people appreciate it!
Wow, well explained. Keep going.
I am glad you liked it
Please I will like to mail you privately. Can I have your mail?
daniel.gyan@yahoo.com
Thanks, super helpful!
Glad it was helpful!
Thank you very much! If we have more than one group, how can we separate their graphs? It gave all of them together and was really confusing...
Put the different data sets on different data sheets
Thanks so much
You're welcome!
nice job, very well explained.
I am glad you liked it
When we apply formulae of dpph
please can you explain your question further?
@@Dr.DanielAddoGyan can you guide me step by step how to use graphpad prism to analyze Dpph result like plant extract absorbance, methanolic , and ascorbic acid activity absorbance.and which formula we used tell me research paper reference.M new in this field
Omg i am in the middle of analysis and i needed this so much. Thanks very much.
You are welcome
Very good explaining, your definitly the cooler Daniel :)
Thanks! 😃
thanks a lot for doing so
I am glad you like it
Indeed nicely explained. Thanks a lot
Most welcome!
Thanks for the useful video.
Glad it was helpful!
very very very clear explanation thanks
I am glad you like it :)
Very Helpful Thanks very very much!!!
Glad it helped!
Very nice tutorial, are you a Liberian?
Thanks. i am Ghanaian
Thanks you sir
I am glad you liked it
Thank you!
Found this video very helpful. Thank you!!
Welcome. Glad it helped. Please subscribe
Well explained
Thanks
Thanks Daniel, it's really helpful.....
Welcome
Thank u so much ,so easy now !! ^^
I am very glad it helped you
THANK YOU SO MUCH!
Glad it helped!
THANK YOU VERY MUCH
Welcome
this really helped a lot. thanks
Thanks. I am glad it helped
my log transform data is negative??? so how i can fix this problem??
Hello. If the value of the concentration is less than 1.0 (for instance 0.01) then the log becomes negative. To solve this you can express the concentrations in another unit that makes the value greater than 1.0. For example a concentration of 0.1 milliMolar can be expressed as 100 microMolar. As a result your IC50 value will be in this new unit.
owhhhhh.... but what if the unit is percentage??? let say 0.5 % (v/v) concentration?
You could consider converting to another unit like parts per million (ppm).
make sense.... thank you so much, Sir...
You are Welcome. Glad i could help
UGH THANK YOU!!!
Welcome
Thanks ...
welcome
Thank you.
thanks.glad you like it
Thank you. Perfect
Welcome
it's amazing. thanks =)
Thanks
+Daniel Addo Gyan 😉🎉🎉🎉
Hello, two questions. if the variables of concentration are presented in nm, the IC50 value in which units gives them to you? and If you use another program like Kaleidragrap, I imagine the value should be the same?
Yes. The units of the IC50 is the same as the unit of the concentration variable
Hey daniel, thank u for the effort...to put up the video...it was really helpful!
Glad it was helpful!