I have two tissues and they are not treated; I would like just to compare the basal level of expression of the gene of interest (goi) in these two types of tissue (tissue1 and tissue2). After qPCR, I have the following result: enter image description here

I wonder how am I supposed to make a decision. Will in this case goi be more expressed in tissue2 ? ctrl - is a housekeeping gene that we use for our types of tissue. Marked in yellow are just mean values. The last column is mean goi_value - mean ctrl_value qPCR quality is not great.


1 Answer 1

  1. You normalized the data to the control and obtained the normalized ct-values. Let's call it Δct.
  2. Now you need to find the relative difference between them. In order to do that, the first step is that you calculate the ΔΔct -> condition_1_normalized - condition_2_normalized.
  3. Calculate the actual difference: condition1/condition2 = 2^-ΔΔct

In your case the answer would be: tissue1/tissue2 = 2^-2.4 = 0.19 The expression of tissue 1 is 0.19 of that in tissue 2. In other words the expression is about 5.2x higher in tissue 2 than in tissue 1.

  • $\begingroup$ thank you, but it's not exactly what I need. I am not looking for a fold change, but just a bare data to put it on a dotplot, for example. Say tissue1 must have some value and tissue2 as well. Not that tissue1 is a "control" and considered as "1" or zero. But it must have some value. Both of them. $\endgroup$
    – Lara
    Aug 21, 2023 at 15:53
  • $\begingroup$ also how did you calculate that 5.2x ? $\endgroup$
    – Lara
    Aug 21, 2023 at 16:34
  • $\begingroup$ I see. If I understand correctly, you want some kind of absolute value for your gene of interest for each of the tissues. Unfortunately, with the data you provided, that is not possible. You can only calculate the relative difference. For absolute quantification you need to run a standard with DNA of known quantity. $\endgroup$
    – Felix H.
    Aug 22, 2023 at 6:53
  • $\begingroup$ The 5.2x is simply the inverse of 0.19 -> 1/0.19 Then instead of tissue1/tissue2 you get tissue2/tissue1. $\endgroup$
    – Felix H.
    Aug 22, 2023 at 6:54

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