Using undergraduate molecular biology labs to discover targets of miRNAs in humans
Idica A, Thompson J, Munk Pedersen I, Kadandale P.
Using your qPCR results (CT method 1)
We use these calculations to use the threshold cycle data obtained by qPCR (the CT values) to determine
change in expression of your candidate gene. Recall that the CT value inversely correlates with the
starting amount of cDNA. A smaller amount of starting cDNA will require more amplification before the
product can be detected.
You must calculate the Delta Delta CT for your target gene and the Positive control using the following
information:
CT = CT(control) - CT(miR)
Where:
CT(control) = CT(target-control) - CT(endogenous control-control)
CT(miR) = CT(target-miR) - CT(endogenous control-miR)
You can then calculate the ratio of expression as 2CT
Remember that higher expression means that your threshold cycle will be lower! Also pay attention to
your –RT readings. What does it mean if they are high? What does it mean if they are low?
Worked example:
miR
Sample
Control
Sample
CT
Endogenous
Positive
Target
CT
14.7492
Endogenous
15.5057
28.1164
Positive
26.7152
28.8212
Target
25.4664
Endogenous
Undetermined
Endogenous
Undetermined
Positive
Undetermined
Positive
Undetermined
Undetermined
Target
Undetermined
Target
(Note: Undetermined = no amplification)
miR
CT(target)
14.072
= 28.8212 – 14.7492 =
CT(positive) = 28.1164 – 14.7492 =
13.3672
Control
CT(target)
9.9607
CT(positivel) = 26.7152 – 15.5057 =
11.2095
CT(target) = 9.9607 – 14.072
= -4.1113
Ratio of expression = 2-4.1113 = 0.056
CT(Positive)
= 11.2095 – 13.3672
= -2.1577
Ratio of expression = 2-2.1577 = 0.224
1
= 25.4664 – 15.5057 =
Adapted from http://blog.mcbryan.co.uk/2013/06/qpcr-normalisation.html
Using undergraduate molecular biology labs to discover targets of miRNAs in humans
Idica A, Thompson J, Munk Pedersen I, Kadandale P.
Therefore Target IS decreased by miR overexpression (positive control worked, shows
decreased expression).