Gene A control null ct is 24.76 and my kd down gene ct is 24.2
GAPDH control 20.7936306 gene 19.23660088.
I am calculating 50% knockdown. but other s considering there is no knockdown based on gene ct values (24.76 and 24.2)
As I know we need to calculate 2^-ct based on loading control. GAPDH will tell if I load more or less.
Please suggest.
Hi Swati,
with the numbers you are indicating you can use the deltaCt method, as you mentioned, or the deltadeltaCt method and as you said it will show a close to 50% reduction in your knockdown vs. control. I would however be a bit hesitant if these were my results as there is a considerable variation in your GAPDH values that would indicate that you used more template in your knockdown samples. With the data you provided is a bit difficult to tell, as you only provided 1 number/condition and I don't know if those are single values, averages of technical replicates, averages of biological replicates, etc. Ideally you would see a difference in the gene "A" without seeing a difference in the housekeeping gene. My recommendation would be to repeat the experiment again if possible with technical and biological replicates making sure that you have the same amount of sample (e.g. lyse the same number of cells, use the same amount of RNA/cDNA, etc). If you still get the same results I would make sure that your particular treatment does not have an effect in the housekeeping gene. You could try some other housekeeping genes that are not affected at all by your treatment so that you can actually used them for normalizing. It could also be that your treatment has an effect in the proliferation or viability of the cells and you should take that into account once you lyse your sample .
Hope this helps
Ezequiel
Hi Swati,
with the numbers you are indicating you can use the deltaCt method, as you mentioned, or the deltadeltaCt method and as you said it will show a close to 50% reduction in your knockdown vs. control. I would however be a bit hesitant if these were my results as there is a considerable variation in your GAPDH values that would indicate that you used more template in your knockdown samples. With the data you provided is a bit difficult to tell, as you only provided 1 number/condition and I don't know if those are single values, averages of technical replicates, averages of biological replicates, etc. Ideally you would see a difference in the gene "A" without seeing a difference in the housekeeping gene. My recommendation would be to repeat the experiment again if possible with technical and biological replicates making sure that you have the same amount of sample (e.g. lyse the same number of cells, use the same amount of RNA/cDNA, etc). If you still get the same results I would make sure that your particular treatment does not have an effect in the housekeeping gene. You could try some other housekeeping genes that are not affected at all by your treatment so that you can actually used them for normalizing. It could also be that your treatment has an effect in the proliferation or viability of the cells and you should take that into account once you lyse your sample .
Hope this helps
Ezequiel
Hi Swati
I agree with Ezequiel, your endogenous has a difference of 1.5 and that is a lot (considering you started with the same amount of RNA and your RT efficiency was the same). If your standard deviation is low I don't think GAPDH is a good control for you. You should check other possibilities.
I would also like to add that you should verify your qPCR efficiency before you use 2-ddCt. If your primers efficiency don't match you should use the Pfaffl method.
Hi
I also agree with the above suggestions on your data. For such small differences you need to be very sure....so include more replicates, check efficiency and if possible go for absolute quantification to be more precise.
bye
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