No abstract

In reverse transcription-quantitative polymerase chain reaction (RT-qPCR) studies, endogenous reference genes are routinely used to normalize the expression of target gene studies. In order to precisely evaluate the relative expression of genes in the cells of mice suffering from Kidney Yang Deficiency Syndrome (KYDS) in response to influenza A virus (IAV) H1N1 using RT-qPCR, it is crucial to identify reliable reference genes. In the present study, 15 candidate reference genes (Actb, β2m, Gapdh, Gusb, Tuba, Grcc10, Eif4h, Rnf187, Nedd8, Ywhae, 18S rRNA, Rpl13, Ubc, Rpl32, and Ppia) were investigated in lung cells from KYDS mice infected with IAV H1N1. NormFinder, BestKeeper, and GeNorm were used to assess the stability of reference genes. The results were authenticated over extended experimental settings by a group of 10 samples. In the present study, we explored a novel method using dual-gene combinations; the difference in gene expression between the model and normal control groups was statistically analyzed by an independent-samples t-test, and the difference in the mean value between the two groups was compared. A P value > 0.05 and the lowest absolute value of the difference indicated the optimal reference two-gene combination. Four additional host innate immune system-related genes (TLR3, TLR4, TLR7, and RIG-I) were analyzed together with the two treatment datasets to confirm the selected reference genes. Our results indicated that none of these 15 candidate reference genes can be used as reference gene individually for relative quantitative fluorescence PCR analysis; however, the combination of Grcc10 and Ppia, based on the process of calculating the higher P value and lower difference values between groups, was the best choice as a reference gene for the lung tissue samples in KYDS mice infected with IAV. This technique may be applied to promote the selection process of the optimal reference gene in other experiments.

Objective. Influenza virus poses a major threat to human health and has serious morbidity and mortality which commonly occurs in high-risk populations. Pharynx and larynx of the upper respiratory tract mucosa is the first defense line against influenza virus infection. However, the ability of the pharynx and larynx organ to eliminate the influenza pathogen is still not clear under different host conditions. Methods. In this study, a mouse model of kidney yang deficiency syndrome (KYDS) was used to mimic high-risk peoples. Two different methods of influenza A (H1N1) virus infection by nasal dropping or tracheal intubation were applied to these mice, which were divided into four groups: normal intubation (NI) group, normal nasal dropping (ND) group, model intubation (MI) group, and model nasal dropping (MD) group. e normal control (NC) group was used as a negative control. Body weight, rectal temperature, and survival rate were observed every day. Histopathologic changes, visceral index, gene expressions of H1N1, cytokine expressions, secretory IgA (SIgA) antibodies of tracheal lavage fluids in the upper respiratory tract, and bronchoalveolar lavage fluids were analyzed by ELISA. Results. e MD group had an earlier serious morbidity and mortality than the others. MI and NI groups became severe only in the 6 th to 7 th day after infection. e index of the lung increased significantly in NI, MI, and MD groups. Conversely, indices of the thymus and spleen increased significantly in NC and ND groups. H&E staining showed severe tissue lesions in MD, MI, and NI groups. H1N1 gene expressions were higher in the MD group compared with the MI group on the 3 rd day; however, the MD group decreased significantly on the 7 th day. IL-6 levels increased remarkably, and SIgA expressions decreased significantly in the MD group compared with the NC group. Conclusions. SIgA secretions are influenced directly by different conditions of the host in the pharynx and larynx in the upper respiratory tract mucosa. In the KYDS virus disease mode, SIgA expressions could be inhibited severely, which leads to serious morbidity and mortality after influenza A virus infection. e SIgA expressions of the pharynx and larynx would be an important target in highrisk populations against the influenza A virus for vaccine or antiviral drugs research.

Kidney-yang deficiency syndrome (KYDS) infected with the influenza virus is a suitable model to imitate a population at high-risk to influenza infection with a high rate of morbidity and mortality. However, the specific molecular mechanisms underlying this disease remain unclear. A stable reference gene is essential as an internal control for molecular biology research of this condition. Reverse-transcription-quantitative PCR (RT-qPCR) is considered an extremely sensitive technique used for absolute and relative quantification of target genes transcript levels. To accurately estimate the relative expression of genes in cells from mice with KYDS in response to infection with influenza A virus subtype H1N1 (A/H1N1) virus using RT-qPCR, it is necessary to identify suitable reference genes. In the present study, analysis of 10 reference genes (Act-β, β2m, GAPDH, Gusβ, Tubα, Grcc10, Eif4h, Rnf187, Nedd8 and Ywhae) was performed across a set of 4 tissue types: Lung; heart; liver; and kidney. KYDS mice were inoculated with A/H1N1 virus or a mock control. For analysis, geNorm, BestKeeper, NormFinder, and Bio-Rad Maestro™ statistical programs were used for the estimation of the stability of the reference genes. The results were authenticated through extended experimental settings using a group of 10 samples, parallel to 3 additional innate immune system-associated genes of the host, TLR3, TLR7 and RIG-I, which were also analyzed using the same algorithms. From the 4 algorithms, taking into account the joint analyses of the ranking order outputs, the 2 genes Ywhae and Nedd8 were identified to be the most stable for mice with KYDS following infection with A/H1N1 virus. In contrast, the least stable genes in all 4 tissues were GAPDH and β2m. These results may affect the choice of reference genes in future studies that use RT-qPCR analysis of target genes in experimental conditions, such as mice with KYDS infected with influenza A virus.

Background Bionovation's CSFA800 is a new automated digital cell imaging analyzer. We evaluated the performance of the CSFA800 by comparing it with artificial peripheral blood white blood cell counting. Methods According to inclusion and exclusion criteria, 131 randomly selected samples (77 abnormal samples and 54 normal samples) were compared. Correlations between automated and manual counting results were analyzed. Manual counting was carried out according to the guidelines of the Association of Clinical and Laboratory Standards. Results Counts of neutrophils, lymphocytes, monocytes, eosinophils, basophils, and immature granulocytes obtained from CSFA800 and artificial methods were linearly and positively correlated, with R values of 0.73, 0.65, 0.24, 0.2, 0.4, and 0.63, respectively, all p < 0.05. Therefore, correlations between CSFA800 and manual counting are acceptable. Compared with the DI‐60 Automated Digital Cell Morphology System (DI‐60; Sysmex), CSFA800 is more efficient and can analyze 20,000 cells in 1 min. However, the overall accuracy of CSFA800 is not as good as DI‐60, although its counting performance is better for basophils. Conclusions The performance of CSFA800 for WBC counts is acceptable, and it displayed good performance for neutrophils, lymphocytes, and immature granulocytes. Compared to DI‐60, CSFA800 is more efficient but has slightly lower overall accuracy. To some extent, CSFA800 is helpful to optimize the clinical laboratory workflow and improve the working efficiency of inspectors.