comparison of mRNA expression - (Oct/09/2007 )

hi all
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

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Personally, I almost always prefer relative comparisons. This eliminates the need for a standard curve.

1. Choose one or two housekeeping genes (i.e. 18S rRNA, Actin, RPL5, etc) and design or order primers for those genes.
2. qPCR all your genes of interest and compare/normalize to your internal control genes.
3. 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.

If you want to be a bit more complicated, you can also determine your primerpair PCR efficiency too (see Pfaffl M, 2002*), but this (in my experience) only provides a modest improvement in most cases.


* A new mathematical model for relative quantification in real-time RT-PCR. Michael W. Pfaffl (2001), Volume 29, Issue 9 e45 [Abstract] [Full text]

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