Chicken embryo fibroblasts (CEFs) are among the most commonly used cells for the study of interactions between chicken hosts and H5N1 avian influenza virus (AIV). In this study, the expression of eleven housekeeping genes typically used for the normalization of quantitative real-time PCR (QPCR) analysis in mammals were compared in CEFs infected with H5N1 AIV to determine the most reliable reference genes in this system. CEFs cultured from 10-day-old SPF chicken embryos were infected with 100 TCID(50) of H5N1 AIV and harvested at 3, 12, 24 and 30 hours post-infection. The expression levels of the eleven reference genes in infected and uninfected CEFs were determined by real-time PCR. Based on expression stability and expression levels, our data suggest that the ribosomal protein L4 (RPL4) and tyrosine 3-monooxygenase tryptophan 5-monooxygenase activation protein zeta polypeptide (YWHAZ) are the best reference genes to use in the study of host cell response to H5N1 AIV infection. However, for the study of replication levels of H5N1 AIV in CEFs, the β-actin gene (ACTB) and the ribosomal protein L4 (RPL4) gene are the best references.

BackgroundThe selection of stably expressed reference genes is a prerequisite when evaluating gene expression, via real-time PCR, in cells in response to viral infections. The objective of our study was to identify suitable reference genes for mRNA expression analysis in chicken embryonic fibroblasts (CEF) after infection with avian leukosis virus subgroup J (ALV-J).FindingsThe expression levels of 11 potential reference genes in CEF infected with ALV-J were determined by real-time PCR. The expression stability of these genes were analyzed and ranked using the geNorm tool. Analysis indicated that the genes RPL30 (ribosomal protein L30) and SDHA (succinate dehydrogenase complex, subunit A) were the most stably expressed genes in the ALV-J infected CEF.ConclusionsThe RPL30 and SDHA were deemed suitable for use as reference genes for real-time PCR analysis of mRNA gene expression during ALV-J infection, whereas commonly used ACTB and GAPDH are unsuitable to be reference genes.

Myasthenia gravis (MG) is an autoimmune neuromuscular disease characterized by the production of antibodies against acetylcholine receptors (AChRs). Qiangji Jianli (QJJL) decoction is an effective traditional Chinese medicine (TCM) that is used to treat MG. Our study aimed to investigate the effect of QJJL decoction on MG and to clarify the mechanism by which QJJL regulates mitochondrial energy metabolism and mitochondrial fusion and fission (MFF). SPF female Lewis rats were administered Rat 97–116 peptides to induce experimental autoimmune myasthenia gravis (EAMG). The treatment groups received QJJL decoction (7.8 g/kg, 15.6 g/kg and 23.4 g/kg). Mitochondria were extracted from gastrocnemius tissue samples to detect respiratory chain complex enzymatic activity. Quantitative PCR and western blot analysis were performed to detect Mfn1/2, Opa1, Drp1 and Fis1 mRNA and protein expression, respectively, in the mitochondria. Transmission electron microscopy examination was performed to show the improvement of mitochondria and myofibrils after QJJL treatment. The results indicated that QJJL decoction may attenuate MG by promoting the enzymatic activity of respiratory chain complexes to improve energy metabolism. Moreover, QJJL decoction increased Mfn1/2, Opa1, Drp1 and Fis1 mRNA and protein expression to exert its curative effect on MFF. Thus, QJJL decoction may be a promising therapy for MG.