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Old 09-08-2014, 11:56 AM   #1
stormin
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Default earliest detectable RNAseq differential expression?

We are trying to determine the best time points to be used for detecting transient transcriptome response after treatment. Are there any studies done prior that looked at the best times for detection? Also when is the earliest detectable response recorded?

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Zach
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Old 09-08-2014, 12:26 PM   #2
dpryan
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I hope you realize that there will be no generally applicable answer to your question.
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Old 09-08-2014, 12:48 PM   #3
stormin
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But there must be a lower limit on this question. For example, 15 minutes post treatment is probably not going to be enough time for new mRNA to accumulate. Now, what about a hour? I just want to see if there are any prior study or personal experience on this. I'm sorry if the original question is confusing.
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Old 09-08-2014, 12:52 PM   #4
dpryan
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15 minutes is probably enough for immediate early genes. There's no definition for transient. You could mean hours or days and even then there'd be no general answer (some changes will only be detectable earlier, others later). Do a pilot experiment with a few samples to have a look at things.
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Old 09-08-2014, 09:32 PM   #5
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Some responses are very fast. For example, my academic lab looked at low oxygen responses and when our samples were exposed to normal oxygen for just a few minutes before freezing in liquid nitrogen we saw expression changes due to reoxygenation. We used a 15 minute exposure to normal oxygen in our experiments as that gave a very robust set of gene expression changes. This was in Drosophila and Drosophila tissue culture.
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Old 09-09-2014, 06:34 AM   #6
mbblack
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Quote:
Originally Posted by stormin View Post
But there must be a lower limit on this question. For example, 15 minutes post treatment is probably not going to be enough time for new mRNA to accumulate. Now, what about a hour? I just want to see if there are any prior study or personal experience on this. I'm sorry if the original question is confusing.
Without knowing anything about the treatment (is this a xenobiotic? - pharmaceutical or industrial chemical?, pesticide?, is it reactive or stable?, is it a small or large molecule?, or is it a dose of radiation or some other "treatment"?), the cell type (I'm assuming you are talking about some sort of in vitro treatment?) the culture conditions and so forth there is no way to even give you reasonable guesses as answers.

Keep in mind that gene expression is the sum of all processes giving rise to a particular concentration of a particular mRNA at any given time. There are small molecules that can have an affect on measured gene expression almost instantly upon exposure. There are xenobiotics which will induce several cascades of expression changes over time from the first few hours to days out from initial exposure. Often your limitation for long term exposures is simple cell viability, but you may detect adaptive changes in gene expression profiles for as long as you can keep the cells alive.

The "best" time for sampling will vary tremendously depending on the specifics of the treatment, and the cell type being exposed. A pilot study is really the only way to pick good exposure(s) and time ponts for your particular treatment and cell type. Relying on results from other studies of different compounds or cell types is really not much better than pure guessing in most instances. Even using IVIVE (In vivo to In vitro extrapolation) to compute cellular exposures from animal studies has not been a guarantee of success in my experience, when working with the identical compound.
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Last edited by mbblack; 09-09-2014 at 06:41 AM.
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