Comparison of RNA isolation procedures for analysis of adult murine brain and spinal cord astrocytes
Comparison of RNA isolation procedures for analysis of adult murine brain and spinal cord astrocytes
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用于分析成年小鼠大脑和脊髓星形胶质细胞的 RNA 分离程序的比较
DOI：
10.1016/j.jneumeth.2019.108545
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发表时间：
2020
影响因子：
3
通讯作者：
Siffrin
中科院分区：
文献类型：
--
作者：
Rosiewicz;Crowley;Kerkering;Alisch;Siffrin
背景细胞群和单细胞的分子分析有助于增进我们对神经系统生理和病理过程的理解。然而，这些方法的局限性在于依赖于针对目标细胞群的温和、高效和特异性的富集程序。特别是，这对于紧密互连的细胞来说是一个挑战，例如星形胶质细胞等中枢神经系统（CNS）内源性细胞。新方法在这里，我们采用了最常见的细胞提取方法之一，即酶促组织消化，然后对单个细胞进行荧光激活细胞分选（FACS）。我们评估了不同的酶/机械组织解离程序并分析了不同的星形胶质细胞谱系转基因模型。此外，我们还比较了脊髓与脑的细胞提取效率。结果Glast-CreERT2x tdTomatofl/flor Aldh1l1-CreERT2x tdTomatofl/的 CNS 组织的酶消化，随后进行 FACS，产生了高度纯化的星形胶质细胞。自动组织消化极大地提高了分离的细胞数量。 Aldh1l1-CreERT2 比 Glast-CreERT2 识别出更多的星形胶质细胞；从大脑中分离产生的数量高于从脊髓中分离的数量。与现有方法的比较我们将酶解/FACS方法的效率和纯度与更现代的程序（包括组织均质化和翻译核糖体亲和纯化（TRAP））进行了比较。结论我们发现这两种方法都会产生高度富集的星形胶质细胞RNA。然而，只有 TRAP 分离才能在单一动物水平上可靠地检测脊髓组织中的 RNA 浓度。根据研究的目的，两种方法各有优缺点，但都可用于星形细胞 RNA 分析。
BackgroundMolecular analyses of cell populations and single cells have been instrumental in the advancement of our understanding of the physiology and pathologic processes of the nervous system. However, the limitation of these methods is the dependence on a gentle, efficient and specific enrichment procedure for the target cell population. In particular, this has been challenging for tightly interconnected cells, for example central nervous system (CNS) endogenous cells such as astrocytes.New methodHere we adopted one of the most common methods of cell extraction, namely, enzymatic tissue digestion followed by fluorescence-activated cell sorting (FACS) of individual cells. We evaluated different enzymatic/mechanical tissue dissociation procedures and analyzed different astrocyte lineage transgenic models. Furthermore, we compared the cell extraction efficiency from spinal cord vs. brain.ResultsEnzymatic digestion of CNS tissue of Glast-CreERT2x tdTomatofl/flor Aldh1l1-CreERT2x tdTomatofl/flfollowed by FACS resulted in highly purified astrocytes. Automated tissue digestion strongly improved the isolated cell numbers. Aldh1l1-CreERT2identified more astrocytes than Glast-CreERT2; isolation from brain yields higher numbers than from spinal cord.Comparison with existing methodsWe compared the efficiency and purity of the enzymatic dissociation/FACS approach with a more modern procedure consisting of tissue homogenization followed by translating ribosome affinity purification (TRAP).ConclusionWe found that both methods result in highly enriched astrocytic RNA. However, only TRAP isolation resulted in reliably detectable RNA concentrations from spinal cord tissue on a single animal level. Depending on the aim of the study both methods have advantages and disadvantages but both are acceptable for astrocytic RNA analysis.
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影响因子：
8.8
作者：
Nectow AR;Moya MV;Ekstrand MI;Mousa A;McGuire KL;Sferrazza CE;Field BC;Rabinowitz GS;Sawicka K;Liang Y;Friedman JM;Heintz N;Schmidt EF
通讯作者：
Schmidt EF
影响因子：
25
作者：
Madisen L;Zwingman TA;Sunkin SM;Oh SW;Zariwala HA;Gu H;Ng LL;Palmiter RD;Hawrylycz MJ;Jones AR;Lein ES;Zeng H
通讯作者：
Zeng H
CNS星形胶质细胞在AldH1L1-Creert2 BAC转基因小鼠中的诱导靶向。
影响因子：
--
作者：
Winchenbach J;Düking T;Berghoff SA;Stumpf SK;Hülsmann S;Nave KA;Saher G
通讯作者：
Saher G
影响因子：
6.2
作者：
Kantzer, Christina G.;Boutin, Camille;Bosio, Andreas
通讯作者：
Bosio, Andreas
影响因子：
4.7
作者：
Michalovicz，Lindsay T.;Kelly，Kimberly A.;O'Callaghan，James P.
通讯作者：
O'Callaghan，James P.