It is generally not recommended to compare qPCR data with RNA-seq data directly, as they are based on different principles and can be influenced by different biases. qPCR measures the amplification of a specific target sequence, while RNA-seq measures the transcript abundance of all genes in a sample. Additionally, there are differences in sample preparation, library preparation, and sequencing platforms that can affect the data generated by each method.

Comparing qPCR CT values and RNA-seq TPM values directly can be problematic because they are based on different scales. qPCR CT values are based on the number of PCR cycles required for the signal to reach a certain threshold, while RNA-seq TPM values represent the relative abundance of a transcript in a sample. Therefore, it is not straightforward to convert between these two values, and it may be difficult to draw meaningful conclusions from such a comparison.

If you want to compare gene expression between qPCR and RNA-seq data, it is generally recommended to use statistical methods that are specifically designed for this purpose, such as correlation analysis or linear regression. Alternatively, you may consider using qPCR to validate specific targets identified by RNA-seq, rather than comparing the data directly.

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Hi Nikolay Klyashtornyy , thank you very much for your explanation.

I understand your advice. One of my goal was to validate the RNA-seq data I have with qPCR data. So in this context, I ran real-time qPCR for the genes of interest and I was able to validate and confirm the presence of target genes in my samples.

However, when it comes to the measurement of the abundance of a particular gene of interest in a tissue/sample, how do I correlate the RNA-sequence analysis data with my qPCR results? In other words, how do I know that a particular gene which showed high TPM value on RNA-seq would also be highly expressed in qPCR experiment?

qPCR is considered the more sensitive and direct analysis. You use the RNA-seq to get a list of "interesting candidate genes" to then investigate using qPCR. You don't need to do any direct comparison to the original RNAseq data.

You can't know that the high TPM from RNA seq will also have high expression during qPCR. That's why you do the experiment.