Sounbuli Khetam, Alekseeva Ludmila A, Sen'kova Aleksandra V, Savin Innokenty A, Zenkova Marina A, Mironova Nadezhda L
Institute of Chemical Biology and Fundamental Medicine SB RAS, Lavrentiev Ave., 8, Novosibirsk 630090, Russia.
Faculty of Natural Sciences, Novosibirsk State University, Pirogova St., 1, Novosibirsk 630090, Russia.
Biomedicines. 2024 Nov 10;12(11):2571. doi: 10.3390/biomedicines12112571.
/: Neutrophils have recently gained significant attention due to their heterogeneity in tumor settings. The gene expression profiles of neutrophils from different tumor types are of great interest. Murine splenic neutrophils reflect the immune status of the organism and could be a source of tumor-associated neutrophils in tumor-bearing mice. However, information about appropriate reference genes for RT-qPCR analysis of murine neutrophils in the literature is lacking. The aim of this study was to identify stably expressed reference genes in murine splenic neutrophils. : Bone marrow- and spleen-derived neutrophils were isolated from healthy C57Bl/6 and CBA/LacSto mice. Spleen-derived neutrophils were isolated from mice with Lewis lung carcinoma (LLC) and drug-resistant lymphosarcoma (RLS). RNA was isolated and used for RT-qPCR analysis of 10 selected reference genes. Analysis of reference gene stability was performed using four different algorithms (BestKeeper, NormFinder, geNorm, ΔCt method), and comprehensive ranking was constructed using RefFinder. : The Ct values for the reference genes were in the range of 16.73-30.83 with the highest expression levels observed for B2m and the lowest for Sdha. Differences in the stability ranking performed by different algorithms were observed; however, the overall ranking of the studied reference genes was as follows, from most to least stably expressed: , , , , , , , , , and . Using or for RT-qPCR data normalization significantly affected the interpretation of target gene expression. : and are recommended reference genes for murine splenic neutrophils regardless of their activation status.
/: 由于中性粒细胞在肿瘤环境中的异质性，最近它们受到了广泛关注。来自不同肿瘤类型的中性粒细胞的基因表达谱备受关注。小鼠脾脏中性粒细胞反映了机体的免疫状态，可能是荷瘤小鼠中肿瘤相关中性粒细胞的来源。然而，文献中缺乏关于用于小鼠中性粒细胞RT-qPCR分析的合适内参基因的信息。本研究的目的是鉴定小鼠脾脏中性粒细胞中稳定表达的内参基因。 : 从健康的C57Bl/6和CBA/LacSto小鼠中分离骨髓和脾脏来源的中性粒细胞。从患有Lewis肺癌（LLC）和耐药性淋巴瘤（RLS）的小鼠中分离脾脏来源的中性粒细胞。提取RNA并用于对10个选定的内参基因进行RT-qPCR分析。使用四种不同算法（BestKeeper、NormFinder、geNorm、ΔCt法）进行内参基因稳定性分析，并使用RefFinder构建综合排名。 : 内参基因的Ct值在16.73 - 30.83范围内，其中B2m表达水平最高，Sdha最低。观察到不同算法进行的稳定性排名存在差异；然而，所研究内参基因的总体排名如下，从表达最稳定到最不稳定： ， ， ， ， ， ， ， ， ， 。使用 或 对RT-qPCR数据进行标准化会显著影响靶基因表达的解释。 : 无论其激活状态如何， 和 都是小鼠脾脏中性粒细胞推荐的内参基因。
