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Experimental qPCR Plate Setup and Analysis
Goals
- Test hypothesis
- Typical plate setup with 2 reference genes and a target gene:
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A
| C1BR1 | C1BR1 | C1BR1 | C1BR1 | C1BR1 | C1BR1 | C1BR1 | C1BR1 | C1BR1 | |
B
| C1BR2 | C1BR2 | C1BR2 | C1BR2 | C1BR2 | C1BR2 | C1BR2 | C1BR2 | C1BR2 | |
C
| C1BR3 | C1BR3 | C1BR3 | C1BR3 | C1BR3 | C1BR3 | C1BR3 | C1BR3 | C1BR3 | |
D
| C1BR4 | C1BR4 | C1BR4 | C1BR4 | C1BR4 | C1BR4 | C1BR4 | C1BR4 | C1BR4 | |
E
| C1BR5 | C1BR5 | C1BR5 | C1BR5 | C1BR5 | C1BR5 | C1BR5 | C1BR5 | C1BR5 | |
F
| C2BR1 | C2BR1 | C2BR1 | C2BR1 | C2BR1 | C2BR1 | C2BR1 | C2BR1 | C2BR1 | |
G
| C2BR2 | C2BR2 | C2BR2 | C2BR2 | C2BR2 | C2BR2 | C2BR2 | C2BR2 | C2BR2 | |
H
| C2BR3 | C2BR3 | C2BR3 | C2BR3 | C2BR3 | C2BR3 | C2BR3 | C2BR3 | C2BR3 | |
I
| C2BR4 | C2BR4 | C2BR4 | C2BR4 | C2BR4 | C2BR4 | C2BR4 | C2BR4 | C2BR4 | |
J
| C2BR5 | C2BR5 | C2BR5 | C2BR5 | C2BR5 | C2BR5 | C2BR5 | C2BR5 | C2BR5 |
Conditions |
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- If you want to be extra safe you should also include a water control for each primer pair.
- The cDNA dilution used for the experimental samples will be those determined from the cDNA Concentration qPCR, and you should use at least your best 2 reference genes from the Reference Gene qPCR
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- If you want to be extra safe you should also include a negative control (usually DIW) for each primer pair.
- The cDNA dilution used for the experimental samples will be those determined from the cDNA Concentration qPCR, and you should use at least your best 2 reference genes from the Reference Gene qPCR.
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What you are looking for
- Technical replicates with a standard deviation below 0.2.
- Outliers; if you have an SD for a technical triplicate higher than 0.2, and there is obviously an outlier, remove it. An example is if you have values of 23.34, 23.35 and 30.22, and the 30.22 amplification trace is clearly poor. If the SD is higher than 0.2 and all three measurements are spread, you must keep all three.
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