AQ vs RQ - (Feb/08/2012 )
Hi, I'm new with qPCR. I have a housekeeping gene and 7 samples of target genes. I have measured my RNA concentration and using 1ug of each of the RNA to synthesize my cDNA. Then I'm going to analyze the effect of my treatment to the target genes (whether they are up-regulated or down-regulated compared with the control) using qPCR. So, I dont know which one should I use, the absolute quantification or relative quantification.
Do I have to create a standard curve first by dilute my samples?
you can create a standard curve from a mix of equal volume of all your cDNA samples (I usually dilute cDNA at 1:4 before this to have enough);
For each gene you pass your standards and diluted samples (I usually dilute samples at 1:20 or 1:60 ). For 1 gene, you have to analyze all of them in the same run. I use a standard curve with 1:4 1:16 1:64 and 1:256 are attribute arbitrary units of 25600 6400 1600 and 400 unit/reaction.
You just have to calculate the ratio Gene W / Housekeeping for each sample. I think that it is a sure way.
Relative expression is also nice only if you have no problem with your reaction and similar efficiency for your target genes and housekeeping genes. with absolute quantification, your standard curve indicates if all is ok.
Can I create a standard curve by running my sample using relative quantification? if so, how because I have tried before this and the machine (ABI 7300) seemed doesn't create it automatically unless if I analyze it using absolute quantification
Relative quantification is based on threshold comparison and i think that the software use it for calculation of gene expression: but all information are normally recorded, but i don't know your software works, I would say that it allows either relative or quantitative quantification, you can also export your ct values and perform the analysis by yourself with excel: to make the curve :report ct on the y axis and log (copie number) on the x axis. use the equation with your sample ct and that's done!
you'll find many documents on the internet from suppliers web sites or ppt from users.