Can I Compare mRNA Levels of Different Genes in qrt PCR Results?

[gravenewworld]

I have 5 different cell lines I'm testing and 2 different genes with GAPDH being used as my housekeeping gene which the mRNA for the 2 different genes are normalized to. People have told me that you can't compare the two different gene mRNA levels to each other, but can only compare the mRNA levels of the same gene across the different cell lines. I really don't understand why, is this true?

 

[Neghentropia]

of course you can compare them

what you can't do is to compare your genes of interest in all your different cells lines whithout referring them to the housekeeping.

 

[gravenewworld]

You can? From what I was told you can't compare two different genes to each other due to the fact that you don't know how efficient each primer is for each gene (makes sense I guess). You can only compare the same gene across multiple cell lines after correcting with a housekeeping gene (in this case GAPDH). I'm doing relative quantitation and not generating any standard curves.

 

[Neghentropia]

oh, well, of course.
if you don't know the primers efficiencies you can't do any quantitation...

anyway if you compare genes on different cells lines you may want to use more than one single housekeeping

 

[LudusRex]

Thank you for sharing your qrt PCR results. I understand your confusion and I would be happy to provide some clarification on this issue.

Firstly, it is important to note that qrt PCR is a highly sensitive and accurate technique for quantifying gene expression. However, there are certain considerations that need to be taken into account when interpreting the results.

In your case, you have used GAPDH as a housekeeping gene to normalize the mRNA levels of the two different genes across the five cell lines. This is a commonly used approach in qrt PCR experiments as it helps to account for any variations in sample preparation and amplification efficiency.

However, it is important to keep in mind that the mRNA levels of different genes cannot be directly compared to each other. This is because the expression levels of each gene can vary significantly depending on factors such as the cell type, experimental conditions, and biological processes. Therefore, it is not appropriate to compare the mRNA levels of different genes to each other.

Instead, the purpose of using a housekeeping gene like GAPDH is to compare the relative expression levels of the same gene across different samples. This allows for a more accurate comparison of gene expression within a specific experimental context. It also helps to control for any technical variations that may arise during the qrt PCR process.

In summary, while it may seem intuitive to compare the mRNA levels of different genes, it is not a valid approach in qrt PCR experiments. The use of a housekeeping gene like GAPDH is essential for accurately interpreting gene expression data. I hope this helps to clarify the issue for you. Keep up the good work with your research!