Hi,

There are several reasons to dilute DNA or cDNA concentrations of soil samples prior to performing qPCR assays. One reason is that if the DNA or cDNA concentration is too high, it can lead to saturation of the PCR reaction and result in unreliable quantitative data. Additionally, high DNA or cDNA concentrations can lead to reduced specificity and increased background noise, which can make it difficult to accurately detect and quantify target genes or transcripts.

When you perform a dilution of the DNA or cDNA samples, you need to calculate the gene/transcript expression based on the dilution factor. The general formula is:

Initial gene/transcript expression copy number = (raw number from qPCR machine) x (dilution factor)

For example, if you dilute the DNA concentration by 5:1, the gene expression copy number would be (raw number from qPCR machine) x 5. This will give you the initial gene/transcript expression copy number before the dilution was performed.

It is important to note that, if you are using a standard curve (cDNA or plasmid) for the quantification, you should to use the same dilution factor for the standard curve and samples in order to get accurate results.

Regards.

Thank you very much for the quick and descriptive reply.

Do you mind if I ask you to give me a bit more explanation regarding your last paragraph? By the way, could you please introduce me a book/any source about the basis of both absolute and relative qPCR principles?

"if you are using a standard curve (cDNA or plasmid) for the quantification, you should to use the same dilution factor for the standard curve and samples in order to get accurate results." Please clarify it.

Best regards,

You generally dilute samples to 1) preserve some DNA/cDNA for later use (repeat testing) and 2) Lessen the concentration of inhibitors. Regarding "if we dilute DNA concentration by 5:1, after getting the raw number from qPCR machine, should we multiply the received number by 5 to reach exact gene expression copy number?", yes you would have to multiply by the dilution factor to get the quantity in the starting tube (this is assuming absolute quantification method via real time PCR). Alternatively, you can also use digital PCR if you have access to get absolute quantification without the use of standards. Some materials for you to look at:

https://www.gene-quantification.de/real-time-pcr-handbook-life-technologies-update-flr.pdf

https://www.gene-quantification.de/strategy.html