comparison of mRNA expression - (Oct/09/2007 )
i want to compare mRNA level among various genes of a particular organism. but i am confused about the strategy to do it by qRT-PCR.
i am thinking as below
suppose i'm using an organism from which i want to compare mRNA level among gene A, B, C, D and E.
but i have 2 different strain where in the No. 1 strain gene A is disrupted and in No. 2 strain B gene is disrupted. now i want to see the mRAN level.
so i am thinking that i
for No. 1 strain
cDNA sample from No. 1
and i want to use primers for gene A, B, C, D and E and standard will be constructed by using the plasmid DNA sample of the respective gene. and finally it will normalize by a HK gene standard.
i cant understand this strategy is effective or not. please help me out.
i use Lightcycler from Roche using syber green
Personally, I almost always prefer relative comparisons. This eliminates the need for a standard curve.
- Choose one or two housekeeping genes (i.e. 18S rRNA, Actin, RPL5, etc) and design or order primers for those genes.
- qPCR all your genes of interest and compare/normalize to your internal control genes.
- Choose one strain as a "reference strain" and another strain as your "experimental strain" and then calculate for fold change of gene expression relative to your reference strain. This is also known as the "delta-delta Ct" method, and assumes an uncorrected value for each primers PCR efficiency.
* A new mathematical model for relative quantification in real-time RT-PCR. Michael W. Pfaffl (2001), Volume 29, Issue 9 e45 [Abstract] [Full text]