Establishing validated RT-qPCR workflow for the analysis of oligodendrocyte gene expression in the developing murine brain.

IF 2.4 3区 生物学 Q3 BIOCHEMISTRY & MOLECULAR BIOLOGY
Biochemistry and Cell Biology Pub Date : 2024-12-01 Epub Date: 2024-08-08 DOI:10.1139/bcb-2024-0088
Samantha Smith, Emma R Swan, Kendra L Furber
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引用次数: 0

Abstract

Myelination is essential for the proper conduction of impulses across neuronal networks. Mature, myelinating glia differentiate from progenitor cells through distinct stages that correspond to oligodendrocyte-specific gene expression markers. Reverse transcription quantiatative PCR (RT-qPCR) is a common technique used to quantify gene expression across cell development; however, a lack of standardization and transparency in methodology may lead to irreproducible data. Here, we have designed and validated RT-qPCR assays for oligodendrocyte genes and reference genes in the developing C57BL6/J mouse brain that align with the MIQE guidelines, including quality controls for primer specificity, temperature dependence, and efficiency. A panel of eight commonly used reference genes was ranked using a series of reference gene stability methods that consistently identified Gapdh, Sdha, Hmbs, Hprt1, and Pgk1 as the top candidates for normalization across brain regions. In the cerebrum, myelin genes peaked in expression at postnatal day 21, which corresponds to the peak of developmental myelination. The gene expression patterns from the brain homogenate were in agreement with previously reported RNA-seq and microarray profiles from oligodendrocyte lineage cells. The validated RT-qPCR assays begin to build a framework for future investigation into the molecular mechanisms that regulate myelination in mouse models of brain development, aging, and disease.

建立有效的 RT-qPCR 工作流程,用于分析发育中鼠脑中少突胶质细胞基因的表达。
髓鞘化对神经元网络冲动的正常传导至关重要。成熟的髓鞘化胶质细胞从祖细胞分化而来,经历了与少突胶质细胞特异性基因表达标记相对应的不同阶段。RT-qPCR 是一种用于量化整个细胞发育过程中基因表达的常用技术;然而,方法缺乏标准化和透明度可能会导致数据不可重复。在这里,我们设计并验证了发育中 C57BL6/J 小鼠大脑中少突胶质细胞基因和参考基因的 RT-qPCR 检测方法,这些方法符合 MIQE 指南,包括引物特异性、温度依赖性和效率的质量控制。使用一系列参考基因稳定性方法对八个常用参考基因进行了排序,结果一致认为 Gapdh、Sdha、Hmbs、Hprt1 和 Pgk1 是各脑区正常化的最佳候选基因。在大脑中,髓鞘基因在出生后第 21 天达到表达峰值,这与发育期髓鞘化的峰值相对应。大脑匀浆的基因表达模式与之前报道的少突胶质细胞系细胞的 RNA-seq 和芯片图谱一致。经过验证的 RT-qPCR 检测方法为今后研究大脑发育、衰老和疾病小鼠模型中调控髓鞘化的分子机制建立了框架。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
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来源期刊
Biochemistry and Cell Biology
Biochemistry and Cell Biology 生物-生化与分子生物学
CiteScore
6.30
自引率
0.00%
发文量
50
审稿时长
6-12 weeks
期刊介绍: Published since 1929, Biochemistry and Cell Biology explores every aspect of general biochemistry and includes up-to-date coverage of experimental research into cellular and molecular biology in eukaryotes, as well as review articles on topics of current interest and notes contributed by recognized international experts. Special issues each year are dedicated to expanding new areas of research in biochemistry and cell biology.
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