pcDNA overexpression - (Feb/27/2013 )

How pcDNA overexpression works? I am told that check the protein level not the message to confirm the overexpression? Why this?

It works via a viral promoter. The true measure of whether or not it is working is to check the protein level - mRNA levels are often but not always correlated to protein levels.

bob1 on Wed Feb 27 19:16:46 2013 said:

Thank you for the quick and clear answer. Highly appreciated.

Now I understand, it has to be confirmed by quantifying protein levels. But, if I read papers where they have measured mRNA expression using qPCR, it is not completely wrong? Cell machinaries still first transcribe information present in pcDNA to mRNA using viral promoter?

Yes, the cell machinery is used to produce the protein from the mRNA transcribed off the plasmid. The problem you have if you use a transfected plasmid like the pcDNA type is that often more than one copy will enter the cell, but we have no way of assessing easily how many have entered any cell (or population of cells), so comparison between transfected mRNA levels is nonsensical. Viral promoters are very very strong and as such give you massive levels of transcript, termed over-expression - so doing qPCR on this is meaningless, unless you are using it to tell you if you have transfected the cells properly, in which case it is often easier and quicker to just do a western or use a tagged plasmid such as a GFP construct to tell you if the plasmid is being expressed, all you should need in these cases is a yes/no answer.

Measurement of normal levels of transcript (i.e. untransfected cells) by qPCR is definitely a valid technique, though there are a few problems with that too, as you will find out if you do more of it. All techniques have some problems though, so it is a matter of comparing the problems/difficulties between each and choosing the one that suits you most.

bob1 on Wed Feb 27 19:57:16 2013 said:

bob1,
once again my sincere thanks for your insightful reply.