If I want to prove that the half-life of my mRNA is low, what experiments should I do? I have the q-PCR data, can I determine the half-life from the data?
I guess you have to perform a sort of "time course" experiment, in order to measure mRNA decay over time. Similarly to proteins, the best way is to interfere with mRNA synthesis to block newly transcribed molecules (this would be the time zero) and analyze over time the relative abundance of already present mRNA. In this manner, you can quantitate in a very efficient and precise way the half-life of your mRNA of interest.
Hi,
in order to determine the half-life of an RNA you have to treat your cells with 10µg/ml ActinomycinD, to inhibit DNA transcription. Then you have to collect your cells at determine time-intervals, depending the half-life of your RNA, and isolate RNA. Afterwards, you could do a RT-qPCR for calculating the amount of your RNA in all samples and calculate the decay rate by using first-order rate kinetics. Take into account that when using an internal control, this RNA should be very stable, as it should not vary within all time-points you have taken.
Hope this helps. Good luck.
Apart from the above mentioned methods, if you have access to radioactive facilities, you could pulse your cells with alpha-P32UTP and then chase it for intended period of time. Counts from isolated mRNAs at different time points vs.time will give a decay trace.
agree to all the answers， it is also useful to use 4su to label RNA，and use ActinomycinD at the same time
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