Therefore, the use of a precise and consistently expressed Treatments, developmental stages or genotypes ( 6, 7). However, the expression of the reference genes may vary based onĭiffering experimental settings, sample species, tissues, Improve the precision and consistency of qPCR ( 4, 5). Standardizing theĮxpression profile and making use of suitable reference genes may Gene expression has to be relatively stable. Quantification is frequently used in numerous studies for analysisĬonsidering the experimental conditions, reference genes areĭesignated as internal controls for which, among different samples, Absolute quantification is notĮxtensively used by most researchers, whereas relative When internal control genes are used as a reference ( 2). Setting up and using a standard curve for relative expressionĬan allow you to accurately estimate absolute quantification fromĭata of target genes can be achieved by relative quantification Measured and compared against the expression level of the target Particular transcript, the input copy number is calculated, whereasįor relative quantification, the expression of a reference gene is (RT-qPCR) is an extremely sensitive method for studying absolute or
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.Īt present, reverse transcription-quantitative PCR In contrast, the least stable genes in all 4 tissues were GAPDH and β2m. 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. 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. For analysis, geNorm, BestKeeper, NormFinder, and Bio‑Rad Maestro ™ statistical programs were used for the estimation of the stability of the reference genes. KYDS mice were inoculated with A/H1N1 virus or a mock control. 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. 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. Reverse‑transcription‑quantitative PCR (RT‑qPCR) is considered an extremely sensitive technique used for absolute and relative quantification of target genes transcript levels. A stable reference gene is essential as an internal control for molecular biology research of this condition. However, the specific molecular mechanisms underlying this disease remain unclear. 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.
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