This calculator takes as input the exported CT values of a Roche LightCycler
and calculates the up and down-regulation of genes between two samples using
the ΔΔ-CT method. In short this method first calculates the normalized
CT time for each sample (by subtracting the gene CT time from the reference
gene CT time). Afterwards these two Δ-CT values are compared by subtracting
the Δ-CT of the altered sample from the Δ-CT value from the non altered
sample. The result is the log2 ratio of the up or down regulation. This value
is then transformed to the actual regulation using 2^-(ΔΔ-CT) and
reported accordingly.
To upload a file from the LightCycler, export the data from the
list of CT values and upload the file below. Those files looks somewhat like
Include Color Pos Name CP Concentration Standard
True 16711680 A1 Sample 1 30.80 0
True 65280 A2 Repl. of Sample 1 31.28 0
True 255 A3 Repl. of Sample 1 30.94 0
True 0 A4 Repl. of Sample 1 30.76 0
True 8405247 A5 Sample 5 29.05 0
True 5337862 A6 Repl. of Sample 5 28.88 0
True 9044500 A7 Repl. of Sample 5 29.31 0
True 8691862 A8 Repl. of Sample 5 29.31 0
The layout of the plates is fixed at the moment. Horizontally
replicates (at any dilution) should be positioned. Each column should
have the same dilution. Vertically (in the rows), specific samples
(treated/untreated) and specific genes should be placed. Once the data
is entered you should specify the sample name and gene name of each
row. Afterwards the up and down regulations are calculated as shown below
Capn3 P3
Row
Gene
name
Sample
name
1
2
3
4
5
6
7
8
9
10
11
12
A
35.96
34.78
34.20
35.51
33.69
35.25
34.81
35.54
33.10
33.47
34.23
36.24
B
34.81
34.60
33.80
33.71
33.20
32.22
32.86
33.19
31.45
32.34
32.86
33.50
C
34.12
33.71
33.44
34.33
33.22
32.66
32.98
33.76
31.73
31.76
32.51
33.35
D
29.54
29.20
29.72
29.91
27.68
27.83
27.83
28.30
26.84
27.08
27.47
28.21
E
29.59
29.00
29.49
29.80
27.61
27.50
27.68
28.27
26.79
27.17
28.04
28.96
F
29.66
28.62
28.60
28.80
27.19
27.26
27.56
28.10
26.77
26.87
27.83
30.17
Calculate
ratio from to The reference/housekeeping gene is
delete this file
Capn3
Ratio: 2.67430992136
down Minimum ratio (68% confidence interval): 3.48137875316
down Maximum ratio (68% confidence interval): 2.05433940476
down Minimum ratio (95% confidence interval): 4.53200951996
down Maximum ratio (95% confidence interval): 1.57809323303
down Contact: werner@yellowcouch.orgRTPCR List