Hi,
Recently, I found that one treatment can decrease the level of one interested protein, but affect neither the mRNA level, nor the degradation rate. It is possible that such treatment may suppress the translation of the mRNA. So I wonder to know how to detect the translational efficiency of the mRNA in mammalian cells, and this possibility coule be tested.
If you want to measure the amount of protein generated from a fixed amount of RNA, you have multiple methods to choose from. Two include running SDS PAGE of the protein generated from the transcript and measuring its amount through densitometry. The second is you add a reporter gene downstream of your regulatory sequences. The amount of reporter gene will be proportional to the amount of translation occurring.
if next gen seq is available do Ribo-seq, if not just pull down polysomes-mRNA complexes and check by qPCR before/after drug. one can also go for polysome fractionation - how many polysomes sit on your mRNA - directly answer your question.
good luck
The translation-efficiency can be presented as a ratio between polysome-associated mRNA and cytosolic mRNA. Thus, you can use a well-established method of polyribosome fractionation using sucrose density gradient. Then, you can purify and quantify cytosolic (from all fractions) and polysomal RNA (from polysomes fractions) .
good luck
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