I have Ct values of my housekeeping gene, and i have ct values of 2 genes of interest. I want to show the level of these genes relative to housekeeping gene and relative to each other. I basically think I have to normalize the genes of interest to my housekeeping gene.
But how to do this, please explain stepwise or in as easy manner as possible as I'm new to this.
hi Yashvi Sharma
You can look at this qRT-PCR analysis format sheet with an example ct value. It comprises replicate value with one housekeeping gene. You can add more replicate value accordingly.
Regards,
Saurabh
The expression level of a gene can be expressed as relative to your housekeeping genes. This is part of the standard approach used in most qRT-PCR analysis.
The difference in Ct values between a gene of interest and a house keeping gene is called the ∆Ct ("delta Ct"). If you are looking for changes in gene expression between two conditions, you simply need to find the difference between the ∆Ct of your control and experimental condition, which is called the ∆∆Ct ("delta delta Ct").
Both ∆Ct and ∆∆Ct represent a difference in the number of PCR cycles, and it is assumed that each cycle doubles the amount of product present. Therefore we can convert the ∆Ct and ∆∆Ct values into a linear relative abundance using 2-∆Ct or 2-∆∆Ct.
This website gives a nice overview of this method: https://toptipbio.com/delta-delta-ct-pcr/
One important thing to note is that the Ct values you use for these calculations should be the average of your technical replicates. However, if you have performed a multiplexed qRT-PCR (in which your house keeping gene and your gene of interest are measured in the same well), then you need to calculate the ∆Ct for each well and then use the average ∆Ct of your technical replicates to calculate the ∆∆Ct values.
You can find good explanation in this link
https://toptipbio.com/delta-delta-ct-pcr/
Yashvi Sharma,
Hi!
Comparing two interest genes are not straightforward. First, your amplification eficiency must be equal between genes and the amplicons must produce the same fluorescence, otherwise you will have a bias. The BEST way to compare diferente interest genes is using ABSOLUTE QUANTIFICATION. In order to do so, you have to buy geneblocks gene fragments (IDT) with your sequence of interest (amplicon) and use this as standard curve (copies/reaction).
https://www.idtdna.com/pages/products/genes-and-gene-fragments/double-stranded-dna-fragments/gblocks-gene-fragments
Best regards,
Jacó
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