Hi,
I can't understand well the use of the normalization for analysing reverse transcriptase real-time pcr data and why you need to use housekeeping genes. Actually my issue is that I can't understand well the meaning of the mathematical formula for the normalization. As far as I understand, I subtract the CT value of the experimental sample to the CT value of the housekeeping gene sample. So, what do I "practically " remove when I make this subtraction ? Why do I need to do it for all the samples I have?
Thanks
Hi Silvia,
The comparative CT method you describe was published in this journal article attached. It has been cited almost 19000 times!
Basically, assuming your PCR efficiency is 2 (your double your DNA every cycle, this is not always the case and should be tested for new primers) your CT values will give you the time it takes for the PCR amplicon level to rise significantly above the background signal. The reason you need to normalize to a housekeeping gene is due to the importance of controlling for the amount of RNA/cDNA placed in each reaction. If you start with more material, it will reach this cycle threshold faster. The expression of a housekeeping gene is constant in all cells (not always the case, you need to choose an appropriate HK gene for your experiment, but that's another story). Since it is constant, you can use it to ensure that each reaction is being compared based on your experiemntal question and is not influenced by how much material you started with.
Essentially, when you are doing the delta CT, you are asking how much longer (or shorter) did this gene of interest take to reach a significant level compared to my housekeeping gene, and the delta delta CT asks how much longer this this gene of interest take to reach significant in each experiment.
I hope that helps!
Jason
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC55695/
Hi Silvia,
The comparative CT method you describe was published in this journal article attached. It has been cited almost 19000 times!
Basically, assuming your PCR efficiency is 2 (your double your DNA every cycle, this is not always the case and should be tested for new primers) your CT values will give you the time it takes for the PCR amplicon level to rise significantly above the background signal. The reason you need to normalize to a housekeeping gene is due to the importance of controlling for the amount of RNA/cDNA placed in each reaction. If you start with more material, it will reach this cycle threshold faster. The expression of a housekeeping gene is constant in all cells (not always the case, you need to choose an appropriate HK gene for your experiment, but that's another story). Since it is constant, you can use it to ensure that each reaction is being compared based on your experiemntal question and is not influenced by how much material you started with.
Essentially, when you are doing the delta CT, you are asking how much longer (or shorter) did this gene of interest take to reach a significant level compared to my housekeeping gene, and the delta delta CT asks how much longer this this gene of interest take to reach significant in each experiment.
I hope that helps!
Jason
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC55695/
- Jason's answer is excellent
- however I will add another salient and crucial point
- different biological replicates will yield different Ct values because of differences in the efficiency of RT due to differences in the purity of RNA
- A less pure sample will result in lower RT efficiency, lower company number of a plethora of RNAs and thus higher Ct values than a cDNA population derived from more pure RNA and/ or a higher mass of total RNA
- Thus when comparing 2 biological replicates you will obtain 2 x different Ct values for a particular gene which does not have anything to do with intrinsic differences in copy number; rather a technical artifact due purity/efficiency differences of RNA/cDNA populations. Crucially howver those differnces are also incurred by other genes like housekeeping genes. In other words although absence Ct values differ between biological replicates the difference between a GOI and housekeeper will not. This in effect is the purpose of normalisation: by correcting or normalisation for such technical arrifacts any resulting differnces between normalised biological replicates are therefore due to differences in copy number or expression and not artefacts
Want to make a short basic answer:
When you put a drop in your well, it might be more or less concentrated in everything. (and your pipet might have some inaccuracy)
If you want to compare your gene expression, it will be biased by concentration of everything (if you have twice more of everything, your RNA will be twice more present)
House keeping genes expression is almost equals (so if concentration of everything is doubled, HKG are doubled too)
So, normalisation by HKG allows you to compare samples without all the bias of total quantity.
Hi Silvia, I can advice you to read this article.
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC55695/
This has been a productive discussion. Thanks to all protagonists
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