qRT-PCR Design

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silverphoenix77
silverphoenix77's picture
qRT-PCR Design

 Hi everyone,

I have > 60 samples to run in qRT-PCR, and I need to determine the gene expression of ~10 genes. Here is my problem, I would like to run my samples in triplicate, but I am not sure what is the best way to setup a 96-well plate. I am working with SYBR green chemistry.

Can I run one gene/plate assuming that their PCR efficiencies are similar?? I know it will be best to run my endogenous control with my target genes, but since I have many samples I am not sure what would be the best solution. Also if my PCR efficiencies are different should I try to find a better housekeeping gene or is there an algorithm that can help solve the problem.

Thank you very much for your time :)
 
 

 

Ivan Delgado
Ivan Delgado's picture
Here are some options you may

Here are some options you may want to consider: 

1. Run as many of your samples as you can in one plate and simply run the rest in other plates. As long as you have the same controls in each plate you should be able to compare the results across plates

2. Do not run triplicates in the same plate. Just run all samples in one plate, and repeat the experiment three times in three different plates.

3. See if you can gain access to a 384-well plate qPCR instrument. This would simply reduce the number of runs you would need to do, and possibly lower the variation in your experiments

Hope this helps,

Ivan

silverphoenix77
silverphoenix77's picture
Thank you very much for your

Thank you very much for your advice, I will probably try option 2 :))