I'm new to PCR, but I want to make sure I'm doing that right thing at that my results will be valid. Let me explain how I'm conducting the analysis.
I have a total of 60 samples that I'm running in triplicate. Therefore I'm dividing that into two separate plates. One plate I'm running the first half and the second plate I'm running the other half. I account for variance I put a control sample in both plates. Since I'm not running all plates on the same day, I think this would be a way to adjust the results. First two plates I did was for GAPDH. I go different values for that control because the threshold (which is set automatically by the machine) was different. In that case, should I change that threshold in the machine based on the first plate, or should I establish a threshold for all my plates, or should I just do it manually on excel adjusting for this difference in the controls? Next I will be running several GOI and I wanted to have this thought through before proceeding.
Thank you.
You should look for a machine able to handle 386 wells plates, it's not good to split your samples in different plates even if you use the same threshold, if you can only use 96 wells plates you will have to split so I would try to do it arranging the same amount of conditions in both, you can compare conditions in each plate but I wouldn't compare conditions between different plates, in other words, it's not good to compare controls samples from one plate with treated samples from another plate, for instance. If you can fit your samples in one plate, removing some of them and performing duplicates instead of triplicates, that would be also correct.
Roberto,
Thank you for the input. We have a limited budget and no one around here has the 386. I understand your point of view! But I will have to figure something else out. Because like I said I have 60 samples and I'm analyzing multiple genes.
José,
Thank you. That was really helpful, and I will look for that software.
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