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Cell & Molecular Biology · Problem type
Fold change from qPCR: ΔΔCt
Use this for “The target’s Ct drops from 27 to 24 while the reference stays at 18. What is the fold change?”
Chart
target: control Ct = 27, treated Ct = 24. reference: control Ct = 18, treated Ct = 18. ΔCt: treated 6, control 9; ΔΔCt = −3, so the fold change is 2−ΔΔCt = 8.
Far below the plateau each curve doubles every cycle: one cycle earlier, twice the start.
ΔCt, treated ΔCttreated = 6
ΔCt, control ΔCtcontrol = 9
ΔΔCt ΔΔCt = −3
Fold change fold = 8
This is an example. Type your own values to start a new problem.
ΔΔCt
=
(
Cttarget,treated
−
Ctref,treated
)
−
(
Cttarget,control
−
Ctref,control
)
fold
=
2
−(
)
ΔCttreated
ΔCt, treated
calculated
ΔCtcontrol
ΔCt, control
calculated
Formulas
ΔCttreated = Cttarget,treated − Ctref,treated
6 = 24 − 18
ΔCtcontrol = Cttarget,control − Ctref,control
9 = 27 − 18
ΔΔCt = ΔCttreated − ΔCtcontrol
−3 = 6 − 9
fold
=
2
−ΔΔCt
8
=
2
−(−3)
Assumptions
•
Both genes double every cycle (100% efficiency), so crossing the threshold one cycle sooner means twice the starting copies.
•
The reference gene is expressed the same in both samples; it corrects for how much RNA was loaded.
•
A fold change above 1 is up-regulation in the treated cells, below 1 down-regulation.
Step-by-step
WE KNOW
Cttarget,treated = 24, Cttarget,control = 27, Ctref,treated = 18, Ctref,control = 18
FIND
ΔCt, treated (ΔCttreated), ΔCt, control (ΔCtcontrol), ΔΔCt (ΔΔCt), fold change (fold)
Step 1 · Find ΔCt, treated (ΔCttreated)
Use ΔCttreated = Cttarget,treated − Ctref,treated
ΔCt is the target’s Ct minus the reference’s, in the treated sample.
ΔCttreated = 24 − 18
ΔCttreated = 6
Step 2 · Find ΔCt, control (ΔCtcontrol)
Use ΔCtcontrol = Cttarget,control − Ctref,control
The same difference in the control sample.
ΔCtcontrol = 27 − 18
ΔCtcontrol = 9
Step 3 · Find ΔΔCt (ΔΔCt)
Use ΔΔCt = ΔCttreated − ΔCtcontrol
ΔΔCt compares the two: treated minus control.
ΔΔCt = 6 − 9
ΔΔCt = −3
Step 4 · Find fold change (fold)
Use fold = 2−ΔΔCt
Each cycle is a doubling, so the fold change is 2 to the power −ΔΔCt; above 1, the target is up.
fold
=
2
−(−3)
fold
=
2
3
fold = 8
Check
Put the numbers back in. Both sides should match.
6 = 24 − 18 ✓
9 = 27 − 18 ✓
−3 = 6 − 9 ✓
8
=
2
−(−3)
✓
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