RNA Isolation - (Mar/24/2011 )

Hello..
Currently m standerdizing my protocol for gene expression analysis of cultured stem cells. I just want to know that why we isolate RNA for gene expression confirmation rather than genomic DNA....

:-)

vidz on Thu Mar 24 08:27:26 2011 said:

Hi.

This is how I understand the things:

Genome or genomic DNA means all coding and non-coding sequences in DNA.

Gene expression is the process by which information from a gene is used in the synthesis of a functional gene product, often a protein. In other words - whcih genes from your genome are swiched on in the moment and giving production.Or more swiched on.

Some genes have a constant expression (or constant production) during the whole life of a cell - these are the housekeeping genes. They are specific for each cell type. Other genes are up-regulated (i.e. become expressed, or swiched on, or productive) in patricular cellular conditions - normal physiological (development, aging, fertilization) or pathological (apoptosis, inflamation, mutations, ets).

When we study gene expression, we usually compare the expression (or production) of a gene of interest toward the expression (or production) of a housekeeping gene, which is accepted as a constant.

Therefore, you need to distinguish only these few particulat genes of interest among all other coding and non-coding sequences, which are sitting in the nucleus. The only one possible way to do this is to follow the mRNAs corresponding to the genes of interest. The fact that you are able to extract a particular mRNA means that its corresponding gene is being expressed (or productive) in the moment of extraction.

In theory - once you extract your mRNA of interest, you generate complementary DNA (cDNA) corresponding to it (thanks to the principle of complementarity), then amplify this cDNA with primers, specific for the gene of your interest and see what happens.

In practice - you extract total RNA and start to fight :-)

Nephrite

The coding DNA is present in all nucleated cells, whereas the coding mRNA is expressed only in those cells that are actively synthesizing the protein. Therefore, if you are interested only in the cells that are active i.e. expressing the RNA, you work with RNA.

Thanks..:-)