I'm working on Penicillium spp. that cause postharvest rot of stone fruit. The project has a gene expression segment where I need to quantify several genes of the pathogen from infected fruit (in vivo). Fruit were at different ripeness levels when inoculated with the Penicillium spp. and incubated for 24h and 48h. Understandably, the riper fruit will be more susceptible and thus lesions will start earlier and develop faster on them compared to less ripe fruit. The pathogen biomass will thus be higher within infected sites (will be isolated for gene expression) of riper fruit. This is my question; what is the importance of a reference gene in this study if it will never be stable across the treatments (ripeness levels) since the infection and colonization of the pathogen will differ?
Hi, Johannes.
I am working on animal cells. So, forgive me if I misunderstood your pathogen-plant work.
What you should check is not absolute expression levels of several target genes, but relative expression levels of them over a reference gene in infected mass during the development of the fruit (ripening). You will be using total RNA from the infected area containing fruit and fungus. If you do not have a reference gene expression level, it would be hard to tell whether you take more fungus or RNA or cDNA from a specific stage of fruit. Therefore, as a control measure in the whole experiments, you check the relative ratios of each gene expression over a reference gene (usually abundant house-keeping gene). Thus, the physical parameters of the infected area and the gene expression ratio will tell the proper comparison whether the several target genes of interest upregulate or downregulate depending on the host developmental stages. The reference gene is considered as an internal control to see proper amounts of RNA or cDNA generated or even loading RT-PCR products on the gel.
This reference gene will allow us to tell which gene is stimulated at different stages of fruit development even if the expression level of house-keeping gene (reference gene) is still consistent. Furthermore it will tell what gene is stimulated at specific development of host.
I agree with Sang Ho Lee. Reference genes provide a common basis to normalize expression levels.
Thank you for the responses.
The work will be done using droplet digital PCR (absolute quantification). I included the common and highly expressed B-Actin as reference gene. The RNA was sent for analysis using the Bioanalyzer and thus input RNA was the same. I thought this would normalize the samples but total RNA means little when both pathogen and host RNA is involved. I also thought quantification (raw data) of the reference gene would be the same over the treatments but this is highly unlikely and thus the reason for the initial question. So I will use a reference gene as an internal control to normalize data? This will eliminate the problem with regards to varying pathogen biomass from samples? Isolating similar pathogen biomasses from infected tissue over the various treatments will be very difficult, if possible. Would B-Actin work for normalization?
I thought you are looking at only pathogen genes. That would bring another reference gene for pathogen, too. You do not know how much of the RNA isolated came from the plant or fungus. Both beta-actin and one abundant fungus-specific gene should be set to normalize both different mass of fungus and plant mass at different stages of infection.
Make a standard mix of fungus and plant mass (maybe 1 vs100 portion),Isolate RNA. RT-PCR. Quantitate or semiquantitate the ratios of fungus and plant mass that corresponding to, for example, the intensity of the PCR product gel bands of the two genes. With this standard mix and two reference gene each, you can sort that out.
If you are looking at plant genes alone forget about fungal reference gene.
- Badges
- Science topic
- Similar topics
- Biological Science
- Genetics
- Gene Expression