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Gene expression varying over time course when it shouldn't (qRT-PCR)

qRT-PCR time-course

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#1 kathy--d

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Posted 12 August 2014 - 06:40 AM

Hi, I'm having a bit of trouble with my experiments so if anyone has any ideas please let me know!

I've been transiently transfecting cells with my gene of interest, waiting 24 hours and then extracting the total RNA and using qRT-PCR to measure it's expression.

 

I'm trying to optimise a time course experiment where I ultimately inhibit transcription using a drug (e.g. DRB) and extract total RNA over a time course to measure the transcript degradation rate of the gene I'm interested in.

 

So far I've been just been trying extracting RNA from cells without transcription inhibition over an 8 hour period, starting 24 hours after transfection (cells have reached100% confluency). I expected that the fold increase (2^ddCt) in expression above endogenous DNA would remain pretty much steady over the time course, but I've been getting wild results.

 

For example, at 0 hours the fold increase was 157, then 60 at 2 hours, 68 at 4 hours, then up to 229 at 8 hours. On other runs I've also gotten weird results that don't remain constant across time.

 

I don't know how to explain this, or what to change in my methods to get steady fold differences across the time points. I don't think it's DNA contamination, as I DNase the samples and check for residual contamination with a standard PCR/gel electrophoresis. I'm also not convinced it's RNA degradation as the bands I get when I run the samples on a gel are pretty good (a bit smudgy though).

 

My only other thoughts are that it might be due to the cell cycle, or because the cells are stressed at 100% confluency and behaving strangely?

 

If anyone has any thoughts please let me know :)



#2 SusieQ

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Posted 12 August 2014 - 11:16 PM

As far as I know, gene expression can vary greatly with time, depending on a wide range of factors such as cell age, cell-cycle stage, overall cell condition, cell medium etc. I'm not sure if the assumption that you will see a stable expression over time is a safe one to make. Usually it's just house-keeping genes that do this.

 

- Are there any other researchers who have looked at the expression of this gene before in transfected cells?

- What kind of gene is it? Which processes is it involved in?

- Are the fold changes you see at timepoint 0, 2, 4 and 8 hours reproducible between experiments?

- Is your amplification specific? Do you see extra bands when you put the samples on gel?

- Suppose the fold increases you see are reliable, and the expression of your gene of interest is just that variable; does incubating your cells with your transcription inhibitory drug produce a dose-dependent response?



#3 Trof

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Posted 13 August 2014 - 06:22 AM

Do you have a selection agent for your gene-containing vector? Because the cells will lose it randomly otherwise. 

 

And as I suppose you don't have the "same" cells analyzed over the timepoints, do you have separate flasks for each timepoint or you take a part of a bigger one?

 

You need to know how stable is the vector in your cells. You may do that by analyzing also the DNA (of cells, plus vector, plus gene) by qPCR measuring the percentage of a cell gene (may be some housekeeping single copy gene, like GAPDH) and some sequence on the vector (possibly part of your gene and part of vector, so you know you are not amplyfing anything that was in the cells).

You may see if the ration remains constant. If so, than the changes in the expression is on the transcription level only (or a problem with a detection, unspecific detection).

 

If the ratio varies, the cells ale losing the vector to transcribe from.


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#4 kathy--d

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Posted 13 August 2014 - 10:01 PM

As far as I know, gene expression can vary greatly with time, depending on a wide range of factors such as cell age, cell-cycle stage, overall cell condition, cell medium etc. I'm not sure if the assumption that you will see a stable expression over time is a safe one to make. Usually it's just house-keeping genes that do this.

 

- Are there any other researchers who have looked at the expression of this gene before in transfected cells?

- What kind of gene is it? Which processes is it involved in?

- Are the fold changes you see at timepoint 0, 2, 4 and 8 hours reproducible between experiments?

- Is your amplification specific? Do you see extra bands when you put the samples on gel?

- Suppose the fold increases you see are reliable, and the expression of your gene of interest is just that variable; does incubating your cells with your transcription inhibitory drug produce a dose-dependent response?

 

 

Hi SusieQ,

 

I've been using ABCE1 and PPPR18 as housekeeping genes (these were selected based on microarray data from Gene Investigator). I've tried this experiment twice so far - the first time the house keeping genes appear to remain constant between the 0, 2, 4 and 8 hour time points. The second time the housekeeping genes showed reduce expression over the time periods. Both times the expression from my gene of interest has varied between time points and is never consistent - e.g. in one experiment it showed a decrease in gene expression from 0-4 hours, and then an increase in expression at 8 hours.

 

The gene that I'm looking at is Zic2 which is a transcription factor. No one else has looked at it's mRNA expression levels before (that we know of).

 

The amplification that we see is specific. The primers have been used by past lab members to amplify the region of Zic2 that I'm investigating.

 

Incubating the cells with DRB doesn't seem to be producing a consistant result either. We've been using 75 mM DRB per well (in a 6 well plate) and this makes one housekeeping gene increase it's expression whilst the other shows a reduction in expression and my gene of interest shows no real change. This concentration was taken from other published papers who also used DRB.

 

We are hoping to see a consistent reduction in the mRNA expression of our Zic2 gene so that we can measure it's decay rate, however our data so far seems to suggest that our expression levels are not consistent even without the DRB added to the cells. Since no one at our institution has performed similar experiments before we're not sure whether we even need the non-DRB time points to be consistent or not in order to accurately measure our decay rate.

 

Any advice that you can give is appreciated!



#5 kathy--d

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Posted 13 August 2014 - 10:26 PM

Do you have a selection agent for your gene-containing vector? Because the cells will lose it randomly otherwise. 

 

And as I suppose you don't have the "same" cells analyzed over the timepoints, do you have separate flasks for each timepoint or you take a part of a bigger one?

 

You need to know how stable is the vector in your cells. You may do that by analyzing also the DNA (of cells, plus vector, plus gene) by qPCR measuring the percentage of a cell gene (may be some housekeeping single copy gene, like GAPDH) and some sequence on the vector (possibly part of your gene and part of vector, so you know you are not amplyfing anything that was in the cells).

You may see if the ration remains constant. If so, than the changes in the expression is on the transcription level only (or a problem with a detection, unspecific detection).

 

If the ratio varies, the cells ale losing the vector to transcribe from.

 

Hi Trof,

 

We haven't been using a selection agent for our construct containing cells. We do have an old stock of antibiotics that we could use but we're wary of it interacting with the DRB that we're using to treat our cells. Since we're only leaving the cells for 24 hours after transfection before we add the DRB, is that enough time for the cells to throw out the plasmid? We leave our transfected cells for 24 hours before performing other experiments such as luciferase assays and have never detected a problem with the plasmid being thrown out.

 

We have separate wells in a 12 well plate that we use at each time point. These all come from the same original flask that is then split between the wells and each well is then transfected individually. We are considering transfecting first and then splitting the transfected cells between the 12 wells but haven't had a chance to test this method yet and see if it helps with the variation in gene expression.

 

In my qRT-PCR I also include 2 housekeeping genes for normalisation. The primers for my gene of interest only bind to my gene of interest, not the vector backbone, but I subtract the dCT value of untransfected cells from the dCT of transfected cells to try and account for the endogenous expression of the gene of interest. Does this method get rid of the need to amplify off the vector backbone as well?

 

Thanks for your help :)



#6 dpo

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Posted 13 August 2014 - 10:41 PM

First thought: Why are you using a plasmid to express your Zic2 when it's also endogenously expressed? Do you want to modify the transcript to see if certain regions are required for its stability?

 

I think the transfection process can have a big influence on the levels you see. I assume that you perform the transfection in different wells and that you then use one well at each time point for lysis and RNA extraction? Differences in transfection efficiency will then also have an influence on the mRNA levels you see in your qPCR. To circumvent this, you can transfect one pool of cells and split them out in different wells the day after. Although you will still get variation, it should be less.

 

You control for the presence of DNA by performing a standard PCR and gel. Why don't you do a qPCR with these -RT samples? This is much more sensitive than the gel you are using.

 

One last thought: you give ratio's for the overexpressed Zic2 vs the endogenous Zic2, but when the endogenous expression is really low (Ct >30), it's very easy to get two-fold differences as you're dividing by a small number that has a lot of intrinsic variation.



#7 kathy--d

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Posted 24 August 2014 - 09:05 PM

First thought: Why are you using a plasmid to express your Zic2 when it's also endogenously expressed? Do you want to modify the transcript to see if certain regions are required for its stability?

 

I think the transfection process can have a big influence on the levels you see. I assume that you perform the transfection in different wells and that you then use one well at each time point for lysis and RNA extraction? Differences in transfection efficiency will then also have an influence on the mRNA levels you see in your qPCR. To circumvent this, you can transfect one pool of cells and split them out in different wells the day after. Although you will still get variation, it should be less.

 

You control for the presence of DNA by performing a standard PCR and gel. Why don't you do a qPCR with these -RT samples? This is much more sensitive than the gel you are using.

 

One last thought: you give ratio's for the overexpressed Zic2 vs the endogenous Zic2, but when the endogenous expression is really low (Ct >30), it's very easy to get two-fold differences as you're dividing by a small number that has a lot of intrinsic variation.

Hi dpo,

 

Sorry for the late reply. That's right, I'm modifying the transcript to examine how different regions impact on stability.

 

I've started transfecting pools of cells and then splitting them into individual wells, so hopefully this will reduce the variation between transfection efficiency. Thanks for the suggestion.

 

I've just been doing the standard PCR to detect for DNA, rather than a -RT control to reduce the costs at this point. I have included this control in my initial attempts at performing this experiment and it looked like the samples were free of DNA. Once I've got the experiments working again and producing good data I will start to include them again.

 

The Ct for the endogenous expression tends to be in the mid-20's. I'm planning to look into different formulas for calculating the fold change from my data, as I know the ddCt has a few drawbacks, such as only using one control gene, and requiring that all the reactions have the same efficiency. So hopefully I'll be able to find something that takes all these things into account.

 

Thanks!







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