I have read that dd-PCR does not require a housekeeping gene - could anyone explain why? We are trying to design a dd-pcr triplex for avian immune gene expression.
Thank you
Ellie
Elizabeth Louise Sheldon
In the ddPCR experiments, the use of reference genes helps to limit the amount of variation that exists between the different samples.
Hello Elizabeth Louise Sheldon
The ddPCR technique shows higher precision and reproducibility. The ddPCR has more advantages than qPCR namely, lower sensitivity to factors that partially inhibit target gene amplification, robustness to variations in PCR efficiency, absolute quantification of the target without requiring a standard curve, and a linearity of the process, which allows the detection of small fold changes in gene expression.
Because of these advantages, the use of a reference gene is not obligatory in ddPCR. However, the application of the reference genes having stable expression may be beneficial for ddPCR data normalization, especially in cases that involve the following conditions:
1) a low number of repeats,
2) the detection of low-fold changes in gene expression, or
3) series data comparisons (such as time-course experiments) in which large sample variation greatly affects the overall gene expression profile and biological interpretation of the data.
Best.
ddPCR (Digital Droplet PCR) is a PCR-based method that partitions the reaction into thousands of tiny droplets, each containing a single copy of the template DNA. The droplets are then analyzed using flow cytometry to determine the number of positive and negative droplets, which in turn reflects the number of copies of the target DNA.
The reason why ddPCR does not require a housekeeping gene is that it is inherently a very sensitive and specific method. The droplet partitioning allows for a high degree of multiplexing, meaning that you can test for multiple genes at once, and the digital nature of the method enables very precise quantification of the target DNA. The high sensitivity of ddPCR means that even low levels of target gene expression can be detected and quantified, which reduces the need for normalization to a housekeeping gene.
That being said, it is always good practice to check for the suitability of the gene of interest as a reference gene and the specificity of the primers, in order to avoid unspecific amplification or cross-reactivity. Additionally, if you are interested in comparing the expression of your target genes across different samples or conditions, it may be useful to include a housekeeping gene as a reference to control for variations in sample quality or RNA integrity.
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