Aladien Geara A reference gene (also known as a housekeeping gene) is commonly used for normalization in real-time PCR investigations because it helps to adjust for fluctuations in the quantity of template DNA or RNA present in the sample. Alternative normalization approaches can be utilized in some circumstances where a reference gene is not available.

The unit mass approach, which includes utilizing a known amount of a bacterial culture as a reference, might be beneficial in your circumstance. This approach may be used to estimate the number of bacteria in a sample, assuming that the number of bacteria is proportionate to the quantity of target DNA present.

Another technique is absolute quantification, which calculates the number of bacteria in a sample based on the amount of target DNA present and the known number of copies of the target gene per bacterium. The use of a standard curve produced from known concentrations of the target DNA is required for this procedure.

It should be noted that these approaches may be less reliable than utilizing a reference gene, and the findings should be evaluated with caution. Furthermore, it is critical to validate the methods utilized and compare them to other ways wherever feasible.

It's worth mentioning that other researchers have used different methods to corroborate their findings, such as Fluorescence In Situ Hybridization (FISH) or qPCR with a set of primers targeting numerous housekeeping genes.