I have a results from qpcr experiment for siRNA after transfection, how to know the effect of siRNA in Knock down from qpcr results
Noha Samir
Let's suppose you have three samples (Control, a sample subjected to the same transfection reagent, siRNA-sample). You have two HKG (G1, G2) and a GOI.
Control-Cq-G1 = 18
Control-Cq-G2 = 18.5
Control-Cq-GOI = 23
Geomean of HKG = 18.25
S2-Cq-G1 = 17
S2-Cq-G2 = 17.5
S2-Cq-GOI = 23.5
Geomean of HKG = 17.25
siRNA-Cq-G1 = 18
siRNA-Cq-G2 = 18.5
siRNA-Cq-GOI = 30
Geomean of HKG = 18.25
Now calculate dCq:
Control-Cq-goi = 23 - 18.25 = 4.75
S2-Cq-goi = 23.5 - 18.25 = 6.25
siRNA-Cq-goi = 30 - 18.25 = 11.75
Some researchers recommend to plot only dCq, if this the case you can plot the last number taking in your consideration the higher number the lower expression.
Note1: I added S2, as it is better to consider the effect of the effect of the transfection reagent and it is worth to say that some researcher add a plasmid to the transfection reagent in sample 2.
Note2: It is common to see higher expression in the supressed sample in comparing with the control one, in this case you have to revise the selectivity of your primer toward the GOI, the time of RNA collection as siRNA can not proceed for long time and assign the place of the primer to the place of siRNA cutting.
Note3: The mentioned method supposed optimized RT-qPCR conditions such primer efficiency, no inhibition ….etc.
Note4: Try to optimize the time of RNA collection and the amount of used siRNA. The ideal time of siRNA to show its effect is between 24 - 48 hours with higher concentration give longer effect.
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