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Experimental qPCR Plate Setup and Analysis

Goals

  • Test hypothesis
  • Typical plate setup with 2 reference genes and a target gene:

1

2

3

4

5

6

7

8

9

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

  • 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

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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Topic revision: r1 - 21 Mar 2019 - 22:01:15 - Main.KateElston
 
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