How to calculate fold change FC, log2FC, Pvalue, Padj, Up and down regulated genes

#rnaseq #logfc #excel
In this video, I have explained how we can calculate FC, log2FC, Pvalue, Padjusted and find Up/down regulated and significant and non-significant genes in RNA seq data using Excel.
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[@natureabioros8686]

Fantastic tutorial. Logically organized and concise!

[@asifmolbio]

Glad you like it

[@karanvirkaushal8386]

Amazing video! So grateful to you sir...

[@asifmolbio]

My pleasure

[@Beautifullife7189]

Thank you sir ,very helpful video ,

[@asifmolbio]

Glad you like it

[@naveenkudupudi]

Thank you for doing this. Just enough detail to understand and do further research.

[@asifmolbio]

Glad if its helpful

[@muhammadwaqasluqman2977]

Thank yiu so much for this video.❤❤

[@asifmolbio]

Glad you like it

[@partha_plethorapedia]

wonderful

[@asifmolbio]

Glad you like it

[@EdehAbigael]

This is very helpful. Thank you

[@asifmolbio]

Glad if its helpful

[@najlaeel8217]

THANK YOU !

[@asifmolbio]

You're welcome!

[@sharonhillary7783]

This video is beyond helpful, even for someone who does not understand excel, thanks a lot sir

[@asifmolbio]

Glad if it’s helping

[@ritziroy148]

Thank you so much! This is so helpful.

[@asifmolbio]

Glad if its helpful

[@syamsidahrahmawati5974]

Thank you very much sir, very clear

[@asifmolbio]

You are welcome

[@Islamicvideos-2666]

Well done dr sahb

[@asifmolbio]

Glad you like it

[@asktayyabrehman]

Thank you sir.

[@asifmolbio]

Thanks

[@Yhusi]

Thanks so much for the detail explanation. If I only want to know the amount expression level of 1 gen in only the control group, should I normalized (devide) with internal control/house keeping gene (like when we calculate in the qPCR analysis)? Should I also log2 it first or use the raw read data? Thank you

[@asifmolbio]

If you have internal control then you can normalize it with that

[@samysolano8526]

Thanks for the video. If I don't have a control group in my experiment and I did ddCt with the difference between my dCt sample value - the highest dCt value in all the samples, what should I use in the first array of the T- test? Thanks

[@asifmolbio]

You can use housekeeping gene CT values as control

[@aqaikalanhassanyar305]

I would like to know when we want to plot the fpkm treated vs control which fpkm should be used in the grsphs?

[@asifmolbio]

Take average of all three, and represent it as mean of expression and then can be shown in the form of graphs

[@mitrabinda1992]

I really appreciate this channel and your quick responses on doubts.. being a beginner, I want to ask that, what are those values from which FC is calculated..

[@asifmolbio]

Thanks, expression value

[@mitrabinda1992]

@@asifmolbio and we make heatmap by using those expression values of three biological replicates?

[@toniromeroconde7193]

Hi Dr. Asif,
If we take into account the FC values ​​to see if there is an up or down regulation. What is the purpose of calculating log2FC?

[@asifmolbio]

Taking the log2 fold change (log2FC) is a common practice in statistical analysis, especially in the context of gene expression data. The log2FC is preferred over the raw fold change (FC) for several reasons:
1. Symmetry: Log transformation makes the fold change symmetric around zero, which simplifies interpretation and statistical analysis.
2. Linear Scale: The log2FC brings the fold change to a linear scale, making it easier to work with in mathematical calculations and statistical models.
3. Stabilization: Log transformation stabilizes variance, helping to satisfy assumptions of normality in many statistical methods.
4. Biological Interpretation: Log2FC is more interpretable in a biological context. For example, a log2FC of 1 means a two-fold change, and a log2FC of 2 means a four-fold change.
By using log2FC, researchers enhance the utility of gene expression data for analysis and interpretation, making it a standard practice in bioinformatics and genomics.

[@pratitiankola8031]

Hi, very nice video and explanation. When I calculate FC I get -Inf values as there are 0 values in my data , so could you please let me know how can I overcome this?

[@asifmolbio]

Thanks ankola, You can search all zeros and delete those rows

[@dr.mohdaamir6688]

How to decide our data is one tailed or two tailed test ??

[@asifmolbio]

Two tailed: Give possibility to see difference on both side at a time (up and down), when you are not sure, which to use, its always better to use two tailed test. (You can also use two tailed when anyone can be control)
One tailed: give possibility to see difference on one side (up or down) only at a time. It is more stringent. (You can also use one tailed when control is already known)
For more details you can watch related videos on CZcams.

[@asifmolbio]

czcams.com/video/i23ZviCIdHw/video.html

[@dr.mohdaamir6688]

@@asifmolbio Thank You so much

[@hendadel8042]

Hi sir. I have data with only 4 subtypes diseases for TNBC, with out normal tissues, How can I do my RNA-Seq analysis?

[@asifmolbio]

Infact always should have a control for comparison. Now you can consider one sub type as control for comparison

[@nwislamicschool]

Excellent Video and you have explained it in such a simplistic manner. My question is if I have Gene expression data on 730 genes in 50 mice with one cancer and 50 mice as control. Can I use this same analogy to determine the significant and non-significant genes based on log 2 data of 50 readings in each group? Highly appreciate your time to respond,

[@asifmolbio]

Yes you can butWhat you mean by 50 readings? Replicates

[@nwislamicschool]

@@asifmolbio Sorry; should have been more clear ; I meant 50 samples

[@asifmolbio]

Yes you can

[@nidaajmal5613]

Hi, this video helped a lot! could you please let me know if there are any databases available online that help in identifying the signaling pathway of the genes that will be filtered out from RNA seq data?

[@asifmolbio]

As such there is no database to identify signalling pathway genes but there are tools which can filter genes and map them to their databases. On my channel you can see video about iDEP RNA seq tool and GO Shiny tool.

[@nidaajmal5613]

@@asifmolbio Thanks I will watch that. There are some tools available for identifying Kegg pathways, like DAVID gene bioinformatic tools, and path Visio, that I found so far.

[@juli1]

Hello, I found that Qiagen Ingenuity Pathway Analysis (IPA) can help you to do so but it's not free. You can search for a free alternative, I'm sure that exists.

[@vahinireddy3707]

Hi sir , thanks for the video!
Can we calculate Pcal from fold change values and also what is the difference between Pcal and padj

[@asifmolbio]

If we comparison then we can calculate p value

[@dr.mohdaamir6688]

Is there any windows based tool to calculate KEGG based Rich factor analysis
in IDEP they are not filtering based on Rich factor

[@asifmolbio]

I think then you need to do it with R. I need to search for online web-based tool, if i got it i will let you know.

[@dr.mohdaamir6688]

@@asifmolbio ok thanks

[@BaNa2C2]

Hi Dr. Asif. Can I know is it okay to set upregulated genes as log2 fold change > 0 and downregulated genes as log2 fold change < 0 ?. I realized most papers use log2 fold change > 1 for upregulated genes and log2 fold change < -1 for downregulated genes . Only few papers set upregulated as log2 fold change > 0 and downregulated as log2 fold change < 0. Thank you

[@asifmolbio]

if you will take logFC >0, it will be still okay. but as you know the level is too low so data would not be much meaningful. So its better if you take FC>1 atleast, would be best if you take FC>2.

[@BaNa2C2]

@@asifmolbio thank you so much Dr.Asif

[@BaNa2C2]

@@asifmolbio another question is if take FC>1 for upregulated then remaining FC1. Is this meant value below 1 to -1 e.g: 0.7, - 0.93 are not included?

[@asifmolbio]

Yes right

[@Sneha_294]

If the control group has some values and the treated cells has 0 as a value, then how can we identify the downregulated genes?
For example, my control groups shows 23, 32, 29 as their read count value. In my treated group all the samples are showing 0. So when I calculate the fold change treated/ control, the fold change is changing to 0. Can you tell me how can we resolve this?

[@asifmolbio]

You should take the value of treated group as 1 for zero. Hopefully will solve the problem

[@Sneha_294]

@@asifmolbio Thank you. Recently binge watching your videos. Very useful!!

[@asifmolbio]

Thats great 😊

[@MuhammadFaizan-mi9yo]

Can you please start a series of videos based on your previous research paper methodology, especially bioinformatics that one would learn strategies of research.and modern bioinformatics tecniques.

[@asifmolbio]

Sure , Don’t forget to check playlists on my channel. Most of the relevant videos are already uploaded.

[@MuhammadFaizan-mi9yo]

@@asifmolbio appricaited sir.

[@MuhammadFaizan-mi9yo]

@@asifmolbioAoa sir how are you? xan you please suggest some paraphrasing and writing tools for Ph.D.? thesis writing paraphrasing,?

[@sangitapaul6463]

May I use this same method to analyze the proteomics data?

[@asifmolbio]

Yes off course

[@sangitapaul6463]

Thank you for your well explained video Sir

[@sangitapaul6463]

Is it possible to do volcano plots with excel?

[@asifmolbio]

@@sangitapaul6463 i will upload a video about volcano soon. Its possible by SR website, not by excel

[@Su.arya31]

Hello sir in geo it is given logFC value , how can we convert to normal fold change value?

[@asifmolbio]

To convert a logFC (logarithm of fold change) value to a FC (fold change) value in Excel, you can use the formula:
=10^(A1)
Assuming your logFC value is in cell A1, this formula will return the FC value. Just replace A1 with the cell reference containing your logFC value.

[@Su.arya31]

Sir thank you for your response , just want to know the given value logFC from database are log base 10 or log base2?

[@asifmolbio]

Base 10

[@BilalRaza-qz7dc]

Hello Can you tell me if i have three groups how i can find FC value

[@asifmolbio]

Take average and follow average

[@jobsworld99]

but you are divided one group to other to find FC value when 3 groups how we will devide..for example i have control, carbon and combine group with each have3 treatments​@@asifmolbio

[@m.awaisahmed8556]

How we can determined either which gene is down or upregulated in case we have more than 2 treatments in Gene expression anlysis ?

[@asifmolbio]

Take an average of all treatments and compare treatment with control one by one

[@m.awaisahmed8556]

@@asifmolbio Thanks Sir .

[@asifmolbio]

Welcome

[@elMARABIYOCHO1989]

Hi. Why did you go for a one-tailed test instead of the two-tailed test? Aren't you trying to see if the genes are upregulated or downregulated? Thanks in advance.

[@asifmolbio]

Two tailed test can also be used if possibility on both side, but i was focusing on only one side

[@elMARABIYOCHO1989]

thank you so much.@@asifmolbio

[@Noor-sr5mj]

please give some practice material

[@asifmolbio]

Sure will upload

[@dr.mohdaamir6688]

I need to convert Gene ID to Gene ontology number. Kindly suggest any direct method to convert or suggest some tools.

[@asifmolbio]

which specie are working with?

[@dr.mohdaamir6688]

@@asifmolbio tomato (Solanum lycopersicum L.)

[@hebanazir4970]

P value give me negative number in calculation

[@asifmolbio]

Then its significant

[@Mr-Ejaz]

hello sir i want to contact you

[@asifmolbio]

Please do an email to [e-mail]