Validating internal controls for quantitative plant gene expression
The data obtained from biological replicates were analysed separately to verify that the variation was not due to the treatment, but was intrinsic to the gene itself.
Nine RGs were ranked in three comparison groups based on their average expression stability (M-value), as shown in Tables 2, 3 and 4.
In fact, plant pathogens, such as viruses, bacteria and fungi, can induce metabolic alterations and gene expression reprogramming in different organs of the host plant, thus modifying the expression of RGs. Proteomic studies revealed the involvement of multiple classes of proteins that are differentially expressed by the plant and the pathogen occurring over a period of weeks after inoculation, as demonstrated by its necrotic or hemibiotrophic phase.
So far, detailed transcriptomic analyses performed during the early stages of plant colonization have not be released, and a selection of RGs for the normalization of q PCR and RT-q PCR gene expression would be useful to standardize and compare the data.
The APX level varied during the experiment time course and according to the inoculum doses, whereas both SOD and CAT resulted down-regulated during the first four days, and up-regulated afterwards, irrespective of inoculum dose. Gene expression analysis is an important tool that is employed to elucidate the complex regulatory networks of the genetic, signalling and metabolic pathway mechanisms that underlie plant-pathogen interactions.
These results can be useful for better elucidating the molecular interaction in the A. Although gene expression microarrays are an ideal tool to provide a snapshot of the global gene transcriptional activity, reverse transcription quantitative real-time polymerase chain reaction (RT-q PCR) assays are normally used to verify results on a smaller scale due to its high sensitivity, high specificity and good reproducibility.
Expression analysis of three target genes, chosen for RGs validation, encoding the reactive oxygen species scavenging enzymes ascorbate peroxidase (APX), superoxide dismutase (SOD) and catalase (CAT) indicated that a combination of stable RGs, such as GAPDH and PP2A, can lead to an accurate quantification of the expression levels of such target genes. actinidiae pathosystem and for RGs selection in bacteria-plant pathosystems.The analyses were performed for three comparison groups considering both low- and high-dose bacterial inocula in the leaves and their combined dataset.In each comparison group, the nine RGs were ranked from the most stable to the least stable.Average E values ranged from 100.7 to 108.2%, with RRT-q PCR was used to quantify the m RNA levels of nine candidate RGs, and the expression stability was investigated.To determine the expression levels of the candidate RGs, the raw quantification cycle (Cq) values were determined.