Plate setup for qRT-PCR

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Urska
Urska's picture
Plate setup for qRT-PCR

 Hi to everyone,

Here is my problem. I have 30 samples equally divided into 3 groups (control (n=10), treatment A (n=10) and treatment B (n=10)). I need to check out expression of 3 different genes, one of them is a reference gene. All my samples will be run in triplicate and here is my dilemma how to setup a 96 -well plate.

I see three options how to run this:
1. run one gene in all samples - one gene/plate
2. run all three genes in one group - one group/plate
3. run all samples and all three genes - one replication/plate

What is the best solution here?

Ivan Delgado
Ivan Delgado's picture
 

 
Hi Urska,

If what you are trying to determine is the expression of your two genes, and your reference gene, relative to each other, then solution #3 is the way to go. You can always run multiple plates of this same experiment to get the necessary replicates.

Now, if you are not interested in looking at the expression of your two genes relative to each other, and what you are really interested in is how each individual gene behaves with your treatments, then solution #1 may be the way to go. Even then, I would encourage you to go with solution #3, but it all depends on what the objective of the experiment is.

Having said this, you could consider a 4th alternative: Break down your groups into 2 sets of 5 samples and run the same experiment as solution #3, but with all the samples in duplicate. This would be similar to running 2 plates of solution #3 (to obtain duplicates), but instead of running all samples at the same time, you would be running 5 samples in one plate (in duplicate) and 5 samples in a second plate (in duplicate). In essence, you would trading one type of variation (variation across-plates for solution #3) for another type of variation (variation across sample groups for solution #4).

Hope this helped. 
Ivan
 

Urska
Urska's picture
 Thank you Ivan for your

 Thank you Ivan for your reply. I'm looking for expression of my genes relative to each other so solution # 3 is the answer.
I did mini test to see if everything is working properly. I took one sample of each group and run all 3 genes. Now I'm wondering if I need to run those 3 samples again with the rest of the samples or not?

Urska

Ivan Delgado
Ivan Delgado's picture
 

 
Hi Urska,

You are welcome. If I were you, I would run all samples, including the 3 you used for the mini test. You can never have enough data, and if you run all samples then you will have a nice control to see if your mini test and your full experiment give you similar results. In other words, running all samples will give you an early indication of how reproducible your experiment is before you run all your samples a second time.

Ivan