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RT-PCR primers on coding sequence or UTRs?


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4 replies to this topic

#1 SF_HK

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Posted 23 March 2009 - 06:06 PM

Hi,

My gene is 2769bp in length and I designed primers for PCR which amplify a region between (forward: 1658-1677bp) and (reverse: 1836bp-1817bp). To check for mRN expression do the pirmers have to be designed within the coding region? are my primers reasonable?

#2 SF_HK

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Posted 23 March 2009 - 06:13 PM

Sorry to clarify the forward and reverse primers amplify a region from 1658bp to 1836bp. The coding region is from 240-1292. Are my prmers reasonable? I get specific bands.

#3 pcrman

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Posted 23 March 2009 - 10:09 PM

So your primers amplify the 3' UTR. Generally speaking, it is fine, but to be on the safe side, I prefer amplifying the coding region. The reason is that according to a recent study from Philip Sharp's group that different 3'UTRs are used when cells respond to environment changes such as stress. In deed, many mRNAs have multiple polyA signals which can be used as a way to evade or allow miRNA regulation.

#4 molgen

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Posted 23 March 2009 - 10:54 PM

It depends on what you want to do, the information you want to get and on the gene.

When I worked on an enzyme that belonged to a gene family I designed the primers inside the coding sequence. I wanted to quantify the expression of all of the family members together. It gave a good correlation to the enzymatic reaction witch doesn't distinguish between the different family members.
Later when I wanted to check under witch condition each member was expressed the primers were designed to the 5'UTR.

So you see, you have to know what you want to do.

#5 SF_HK

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Posted 31 March 2009 - 09:56 PM

It depends on what you want to do, the information you want to get and on the gene.

When I worked on an enzyme that belonged to a gene family I designed the primers inside the coding sequence. I wanted to quantify the expression of all of the family members together. It gave a good correlation to the enzymatic reaction witch doesn't distinguish between the different family members.
Later when I wanted to check under witch condition each member was expressed the primers were designed to the 5'UTR.

So you see, you have to know what you want to do.


I just want to check for the expression of a particular gene in a panel of cell lines my RT-PCR and qPCR.




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