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Livak / Delta Delta Ct qPCR Calculator (editable Ct/Cq values)
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Students edit only the yellow Ct/Cq input cells. All averages, Delta Ct, Delta Delta Ct, and fold change are calculated with embedded Excel formulas.
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GroupSampleHK Ct1HK Ct2HK Ct3HK Avg CtTarget Ct1Target Ct2Target Ct3Target Avg CtDelta CtAverage Control ΔCt (Calibrator)Delta Delta CtFold Change (2^-DDCt)Notes
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ControlControl 117.1917.3417.1617.2330.4530.6630.6530.5913.3613.46-0.111.08Calibrator group
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ControlControl 216.9616.9016.9516.9430.4330.2230.6630.4413.5013.460.040.98Calibrator group
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ControlControl 317.0717.3017.1517.1730.7430.5530.8430.7113.5413.460.070.95Calibrator group
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TreatedTreated 118.0417.9917.9517.9925.8025.7725.8525.817.8113.46-5.6550.25Experimental group
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TreatedTreated 217.9918.0117.9117.9725.5925.7725.6625.677.7013.46-5.7654.23Experimental group
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TreatedTreated 317.9017.6617.8617.8125.6525.5025.7225.627.8213.46-5.6550.14Experimental group
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The yellow cells are technical replications
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Class summary
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Average Control Delta Ct13.46
This is the calibrator
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Average Control Fold Change
1.00
Should be close to 1
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Average Treated Fold Change
51.54Main result
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Interpretation
The treated group is about 51.5-fold relative to the control group.
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Visible classroom protocol
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1. Enter qPCR Cq/Ct values for the housekeeping gene and target gene in the yellow cells. In this file, each sample has two technical replicates: Ct1 and Ct2.
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2. Average the technical replicates for each gene. HK Avg Ct = AVERAGE(HK Ct1, HK Ct2). Target Avg Ct = AVERAGE(Target Ct1, Target Ct2).
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3. Normalize the target gene to the housekeeping gene for each sample. Delta Ct = Target Avg Ct - HK Avg Ct.
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4. Choose the control group as the calibrator. This spreadsheet calculates Calibrator Avg Delta Ct as the average Delta Ct of all rows labeled Control.
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5. Compare every sample to the calibrator. Delta Delta Ct = sample Delta Ct - Calibrator Avg Delta Ct.
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6. Convert Delta Delta Ct to relative expression. Fold Change = 2^(-Delta Delta Ct).
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7. Interpret values. A fold change of 1 means no change relative to control; greater than 1 means up-regulation; less than 1 means down-regulation.
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8. Teaching reminder: the Livak method assumes target and housekeeping PCR efficiencies are approximately equal and close to 100%.
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