Body: <p>Question: Why is there a minus in the <code>2ˆ(–delta delta CT)</code> formula?</p>
  <p>My line of thoughs:</p>
  <p>Consider <code>Ct</code> values of some pPCR experiment (some gene of interest X and a reference gene REF is measured under some experiment condition and a control condition).</p>
  <ol>
  <li><p>The delta CT (<code>dCt</code>) would correspond to normalized <s>expression</s> Ct of X against REF of both conditions. (dCt = X-REF)</p>
  </li>
  <li><p>The delta delta CT (<code>ddCt</code>) would then correspond the the difference of the dCt of the experiment condition minus the dCt of the control condition.</p>
  </li>
  </ol>
  <p>=&gt; If ddCT &gt; 0 then the gene of interest is <s>upregulated</s> downregulated (Ct is antiproportional to gene expression). (the difference of the dCt in the experiment condition is larger than in the control condition)</p>
  <p>But now: the ratio <code>R = 2ˆ(–delta delta CT)</code> would then correspond to a value smaller than 1 (because of the minus).</p>
  <p>I read sentences like</p>
  <blockquote>
  <p>&quot;We see a 10% decrease of expression (R=0.9)...&quot;</p>
  </blockquote>
  <p><s>But actually the raw data clearly shows a upregulation</s>... This is confusing since only the ratio R decreases by 10% but the this would correspond to an increase of raw expression...</p>
  <p><s>I could interprete R as the ratio of the control against the gene of interest. But why not the other way around?</s></p>
  <p><strong>Edit: Ct is antiproportional to gene expression =&gt; dCt does not directly reflect the gene expression.</strong></p>
  <blockquote>
  <p>Sources:</p>
  <p><a href="https://bmcbioinformatics.biomedcentral.com/articles/10.1186/s12859-020-03696-y" rel="nofollow noreferrer">https://bmcbioinformatics.biomedcentral.com/articles/10.1186/s12859-020-03696-y</a></p>
  <p>&quot;We can predict that a plant with a hormone concentration of
  8.85 pg/mL would have an expression of Gene G that is 27% (̂ =0.73) lower than that of plants with average hormone concentration&quot; (just
  below equation 8)</p>
  <p>But clearly in Fig. 3a you can see that the expression at 8.85 is
  obviously higher than with average hormone concentration (x0 = 9.85)</p>
  </blockquote>
  
Comments.ContentLicense: CC BY-SA 4.0
Comments.CreationDate: 2020-11-25T02:55:04.487Z
Comments.Text: Perhaps this pdf of the ddCt package helps: https://bioconductor.org/packages/release/bioc/vignettes/ddCt/inst/doc/rtPCR.pdf . See equations 6, 7 and 8.
Comments.UserDisplayName: Peter
Comments.ContentLicense: CC BY-SA 4.0
Comments.CreationDate: 2020-11-26T14:29:19.503Z
Comments.Text: ok I found the reason: Ct is antiproportional to the gene expression. Therefore the statements from the paper are correct. But thank you for your link, very nice derivation
Comments.UserDisplayName: Paul
Tags: <normalization><qpcr>
Title: Why is there a minus in the 2ˆ(–delta delta CT) method (qPCR)
ThreadText: # Why is there a minus in the 2ˆ(–delta delta CT) method (qPCR)
  
  (Asked by: username_0 on 2020-11-09)
  
  Question: Why is there a minus in the `2ˆ(–delta delta CT)` formula?
  
  My line of thoughs:
  
  Consider `Ct` values of some pPCR experiment (some gene of interest X and a reference gene REF is measured under some experiment condition and a control condition).
  
  1. The delta CT (`dCt`) would correspond to normalized ~~expression~~ Ct of X against REF of both conditions. (dCt = X-REF)
  2. The delta delta CT (`ddCt`) would then correspond the the difference of the dCt of the experiment condition minus the dCt of the control condition.
  
  => If ddCT > 0 then the gene of interest is ~~upregulated~~ downregulated (Ct is antiproportional to gene expression). (the difference of the dCt in the experiment condition is larger than in the control condition)
  
  But now: the ratio `R = 2ˆ(–delta delta CT)` would then correspond to a value smaller than 1 (because of the minus).
  
  I read sentences like
  
  > "We see a 10% decrease of expression (R=0.9)..."
  
  ~~But actually the raw data clearly shows a upregulation~~... This is confusing since only the ratio R decreases by 10% but the this would correspond to an increase of raw expression...
  
  ~~I could interprete R as the ratio of the control against the gene of interest. But why not the other way around?~~
  
  **Edit: Ct is antiproportional to gene expression => dCt does not directly reflect the gene expression.**
  
  > Sources:
  >
  > <https://bmcbioinformatics.biomedcentral.com/articles/10.1186/s12859-020-03696-y>
  >
  > "We can predict that a plant with a hormone concentration of
  > 8.85 pg/mL would have an expression of Gene G that is 27% (̂ =0.73) lower than that of plants with average hormone concentration" (just
  > below equation 8)
  >
  > But clearly in Fig. 3a you can see that the expression at 8.85 is
  > obviously higher than with average hormone concentration (x0 = 9.85)
  
  **Tags:** normalization, qpcr
  
  > **Comments:**
  > **username_1**: Perhaps this pdf of the ddCt package helps: https://bioconductor.org/packages/release/bioc/vignettes/ddCt/inst/doc/rtPCR.pdf . See equations 6, 7 and 8.  
  > **username_0**: ok I found the reason: Ct is antiproportional to the gene expression. Therefore the statements from the paper are correct. But thank you for your link, very nice derivation  
  
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  ## 0 answers
  
  