Question: How to calculate Log2 Fold Change for normalised qPCR data?
gravatar for Genosa
4.1 years ago by
Genosa100 wrote:

Sorry if this is a rather basic question. I am trying to find a good solution on how to calculate fold change for qPCR data normalised to housekeepting genes across different conditions.

For example, I have this data: (CT value) Baseline condition Housekeeping gene: 30.7, 30.6, 30.6 Baseline condition Gene X: 33.0, 34.2, 33.5

Treatment condition Housekeping gene: 31.1, 31.4, 31.3 Treatment condition Gene X: 29.1, 29.4, 29.6

After I normalise the baseline condition gene X / Treatment condition gene X to the housekeeping gene by delta-delta CT, what is the correct formula to calculate log2 fold change of treatment over baseline condition please?

Thank you

qpcr • 18k views
ADD COMMENTlink modified 2.4 years ago by Kevin Blighe69k • written 4.1 years ago by Genosa100
gravatar for VHahaut
4.1 years ago by
VHahaut1.1k wrote:

You can find plenty of nice explanation online (you should consider reading the blog post below):

Computing fold change values for RT-PCR

ADD COMMENTlink written 4.1 years ago by VHahaut1.1k

Got it. Thanks very much

ADD REPLYlink written 4.0 years ago by Genosa100
gravatar for Kevin Blighe
2.4 years ago by
Kevin Blighe69k
Republic of Ireland
Kevin Blighe69k wrote:

Also an answer here, which corroborates with that given in: Computing fold change values for RT-PCR

The standard is the delta delta Ct (ddCt) method, whereby the Ct values in each sample are normalised to:

  1. a housekeeper gene (or genes) within the same sample
  2. the respective values in a control DNA sample

The final, normalised Ct, the delta delta Ct, is then typically put as a negative power to 2:

2 ^ (- ddCt)

For example, we have the following raw Ct values:

                  SampleRep1 SampleRep2 SampleRep3 | ControlDNA1 ControlDNA2 ControlDNA3
Gene1             19         20         19         | 21          21          20
HousekeeperGene   22         22         22         | 21.5        21          22

Gene1, Delta Ct in Sample = [(19+20+19) / 3] - [(22+22+22) / 3] = -2.667

Gene1, Delta Ct in Control DNA = [(21+21+20) / 3] - [(21.5+21+22) / 3] = -0.8333

Gene1, Delta Delta Ct = -2.667 - (-0.8333) = -1.8337

2 ^ (- ddCt) = 2 ^ (- (-1.8337)) = 3.57 fold increased expression of gene1 in sample1

[from: A: How to report and plot qPCR data ]


ADD COMMENTlink written 2.4 years ago by Kevin Blighe69k
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