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actinomycin

measue of mRNA decay

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#1 SF_HK

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Posted 27 February 2019 - 08:20 AM

My drug is increasing proliferation of gene of interest (GOI).  I would like to demonstrate the effect of this drug at the transcriptional level.  

 

Im planning to check if it delays mRNA degradation of my GOI. Has anyone used actinomycin D and correct if Im wrong in how to use it:

 

1. treat cells with actinomycin D for various time points

2. measure mRNA of GOI by qPCR

3. Also detect GOI by protein

4. To ensure actiomycin D is working, I check cyclin D1 mRNA levels after actiomycin D treatment?

 

Many thanks



#2 SF_HK

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Posted 27 February 2019 - 08:42 AM

also, how do we calculate the mRNA half life? for qPCR I would normalise the expression of GOI with actin or GAPDH internal control



#3 bob1

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Posted 28 February 2019 - 09:20 AM

That looks like it should work. You would calculate the half-life by plotting a decay curve, just as you would for any half-life calculation. 

 

Basically you would plot a normalized Ct value over time and calculate at which point 50% of the mRNA was left.






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