My question is regarding the design of time course experiment. I wanted to know the expression level of one miRNAs in H1N1 infected A549 Cells at 24, 48, 72 hrs post infection and to compare it with the non infected cells. I will do the experiment in 12 well plate. I am a bit confused whether I design the experiment that I have 3 wells as non infected cells ( no H1N1 added but only cells) and the other other 3 time points in replicates then I will have 9 wells infected with virus and I will collect them at each time point. then in this case I will compare each time points with the 0 point as a control for all times. The other way is to create a 3 wells as non infected fr each time points meanings that i will have 9 wells as non infected (each 3 wells will be compared with each 3 wells from the 3 time points) and other 9 wells as an infected. then I will compare the 3 replicates of each time points with its 3 well non infected replicate.
What is the right way?
Salam Mohamed,
It is better to compare your result with control for each time, that mean u choose the second way according to your question. Because the expression of miRNA can be also affected with the time.
Good luck.
I agree with Bashir Yousef ...I also use to plan the second way for my experiments ..
Hello Samir,
I understand that you have 12 well plates at your disposal, but 6 wells will be preferably better in this case. That way for each time frame, you will have 3 infected and three control withdrawn once and analysed together. You can run a t-test per time frame with control each time and a one way ANOVA across the three time frames.
Best of luck!
Thanks so much but if I have 3 0 points , each for each time points. which one of them is considered the 0 point for me because finally you need to show the level of miRNAs at 0 time and the level of miRNAs is differ in the all 3 since the 0 point cells left for 24 hours will express miRNAs differentilly if u left them 48 hours.
What is the disadvantages of the first way
Its quite easy,you can seed an extra plate to harvest at 0 hr. I presume your viral absorption will be between 30mins to 1hr,which means 0 hr will be immediatly after viral adsorption.
Your design has an inconvinience of exposing the cells to risk of contermination everytime since they are seeded in the same plate and withrawn multiple times for harvest,besides for statiscical accuracy you need at least three replica for each time frame and control.
May be you got me wrong. 0 point for me is non infected cells ( the basal level of miRNAs without infection) not after virus absorbtion. Now, do you agree with me that defining a triplicate of non infected cells (control) to each time point point without having an extra 3 control wells is wrong in my case since I need to show the control or the level of miRNAs in the non infected state ? ? ?
I agree with Yusuf. You will need time control or non-infected control (whatever you like to call it). Otherwise you might have loose your focus point- whether your miRNA expression changed due to time or due to viral infection. First you can finish up this experiment and then you can decide the best way to explain it or publishing it. But time control is the best way to explain critical things like protein (receptors, enzymes), RNA expression.
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