You need to quantify expression of the INS gene on several stem cell derived ins
ID: 194184 • Letter: Y
Question
You need to quantify expression of the INS gene on several stem cell derived insulin- secreting cell products (SCb) that you have generated: SCb1, SCb2, SCb3, SCb4, SCb5, SCb7 and choose the product that displays the highest INS expression. You extract total mRNA from all your seven cell products, then you generate cDNA by reverse transcription and run real time PCR and you get the following plot.
a. Make a table with the Ct for each sample.
b. Assuming that all seven samples had a Ct of the endogenous gene of 16 and a Ct of the INS gene for the control cells (islets) of 10, express the expression of the INS gene for each cell product relative to islets.
14000 12000 1 0000 O 8000 02 6000 2 4000 0 2000 0 SCb4 CO CO 4000 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 CyclesExplanation / Answer
Ct in Real Time PCR experiment is the number of cycle require for the fluorescent signal to cross the threshold. It is cycle value when the fluorescence point meets the threshold. Ct is inversely proportional to the amount of DNA in the sample. Threshold is the black line in the PCR graph.
a) Approximate values are calculated, as it is not possible to get the decimal places in the graph.
Sample
Ct
SCb1
18
SCb2
25
SCb3
28
SCb4
14.5
SCb5
21
SCb6
35
SCb7
31.5
b) Ct of endogenous sample (reference sample) is 16. INS gene in control cells had a Ct of 10.
DCt is delta Ct
Relative Expression=
D Ct (Islets) = Ct of INS gene in islets-Ct of the endogenous gene for islets
= 10-16=-6
We assume that the Ct value for the endogenous gene in the control islets cells is also 16, as equal amounts of samples are added for control and test samples in Real time-PCR. The endogenous control will not change in control and test samples, as these are housekeeping genes and act as a reference for amount of DNA added.
D Ct (SCb1sample) = Ct of INS gene in SCb1 sample-Ct of the endogenous gene in SCb1 sample
= 18-16=2
D Ct (SCb2sample) = Ct of INS gene in SCb2 sample-Ct of the endogenous gene in SCb2 sample
= 25-16=9
D Ct (SCb3sample) = Ct of INS gene in SCb3 sample-Ct of the endogenous gene in SCb3 sample
= 28-16=12
D Ct (SCb4sample) = Ct of INS gene in SCb4 sample-Ct of the endogenous gene in SCb4 sample
= 14.5.-16=-1.5
D Ct (SCb5sample) = Ct of INS gene in SCb5 sample-Ct of the endogenous gene in SCb5 sample
= 21-16=5
D Ct (SCb6sample) = Ct of INS gene in SCb6 sample-Ct of the endogenous gene in SCb6 sample
= 35-16=19
D Ct (SCb7sample) = Ct of INS gene in SCb7 sample-Ct of the endogenous gene in SCb7 sample
= 31.5-16=15.5
Formula for DDCt or Delta Delta Ct= D Ct of test sample- D Ct of islets
Formula for Relative fold Change is
R= 2^-DDCt
Applying the formulas,
Sample
Delta CT
Delta Delta Ct
Relative Fold change
Islets
-6
SCb1
2
8
0.003906
SCb2
9
15
3.05 x 10-5
SCb3
12
18
3.81 X 10-6
SCb4
-1.5
4.5
0.044194
SCb5
5
11
0.000488
SCb6
19
25
2.98 X 10-8
SCb7
15.5
21.5
3.37 x 10-7
The calculations are in logarithm base 2. Hence, twice the amount of DNA, the Ct values decrease by 1.
Relative fold change is the expression of INS gene product relative to islets.
Sample
Ct
SCb1
18
SCb2
25
SCb3
28
SCb4
14.5
SCb5
21
SCb6
35
SCb7
31.5
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