Q46175364
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Insect larvae develop in decaying organic matter and their defence against various microorganisms must therefore be highly efficient. In the present study, we explored the transcriptional kinetics and induction levels of eight genes in Sarcophaga bullata larvae after infection or aseptic injury. Using real-time PCR, we studied the time-dependent immune response of larvae of the fleshfly S. bullata. We compared the mRNA levels of eight selected genes in induced and non-induced larvae. The third-instar larvae of S. bullata were induced by injecting a bacterial suspension of Escherichia coli, Staphylococcus aureus or Pseudomonas aeruginosa, or by simple aseptic injury with an entomological pin. We used intact larvae as a control for basal mRNA expression. Total RNA was isolated from the whole body, fat body and haemocytes. We determined the mRNA levels of genes encoding sapecin, transferrin, prophenoloxidase 1 and 2, storage-binding protein, cathe psin L, sarcocystatin, and 26/29 kDa protease. We found that there was massive up-regulation of genes encoding the fleshfly peptide sapecin, as well as the protein transferrin. We also detected down-regulation of, or no change in, the expression of genes that encode prophenoloxidase 1 and 2, storage-binding protein, cathepsin L, sarcocystatin, and 26/29 kDa protease.
- MeSH
- Diptera genetika imunologie mikrobiologie MeSH
- Escherichia coli imunologie MeSH
- financování organizované MeSH
- larva genetika imunologie mikrobiologie MeSH
- polymerázová řetězová reakce s reverzní transkripcí MeSH
- Pseudomonas aeruginosa imunologie MeSH
- regulace genové exprese MeSH
- Staphylococcus imunologie MeSH
- zvířata MeSH
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- zvířata MeSH
Cell differentiation depends mainly on specific mRNA expression. To quantify the expression of a particular gene, the normalisation with respect to the expression of a reference gene is carried out. This is based on the assumption that the expression of the reference gene is constant during development, in different cells or tissues or after treatment. Xenopus laevis studies have frequently used eEF-1 alpha, GAPDH, ODC, L8, and H4 as reference genes. The aim of this work was to examine, by real-time RT-PCR, the expression profiles of the above-mentioned five reference genes during early development of X. laevis. It is shown that their expression profiles vary greatly during X. laevis development. The developmental changes of mRNA expression can thus significantly compromise the relative mRNA quantification based on these reference genes, when different developmental stages are to be compared. The normalisation against total RNA is recommended instead.
