Transcriptome-focused research has come a long way since the introduction of the northern blot in the 1970s followed by the quantitative polymerase chain reaction (qPCR) and microarray technology. In recent years, the development of next generation sequencing (NGS)-based techniques has revolutionized the way scientists analyze gene expression and regulation. RNA sequencing (RNA-seq) uses NGS to detect, quantify, and profile the transcriptome. In comparison with older methods, RNA-seq offers better resolution and coverage and, crucially, does not require prior knowledge of the analyzed transcriptome. This opens the door for more从头discoveries. These advancements are allowing scientists not only to quantify gene expression, but to discover new transcripts, alternative splicing sites, and alleles¹。RNA-Seq的广泛使用有助于削减设备成本，使得该分析高度易于访问，并在包括神经科学在内的几乎每个科学和医学领域都带来了突破性的发现。

神经科学方面的科学进步非常需要这种技术来解决科学家在人体中最复杂的器官时遇到的局限性。此外，尽管神经系统具有人体任何系统的细胞异质性最高，但这种细胞多样性的很大一部分和长距离连通性仍然在很大程度上未被发现。克服这些约束依赖于高通量，精确技术的开发和实施，例如适合神经科学的RNA-Seq。

这种技术进步的长期目标是更好地了解大脑的生理环境，介绍其转录组，并将其链接到大脑的独特生理学。在健康环境中识别这些机制将加快与疾病相关的表型和生物标志物的启示，并支持神经病理学的药物发现。比利时罗马尼亚比分直播

在此列表中，我们探讨了神经科学中使用的RNA-seq技术的主要方向及其对该领域的贡献。

1。Single-cell RNA sequencing (scRNA-seq)

解决大脑细胞异质性的问题需要在单细胞水平上探索系统。顾名思义，单细胞RNA测序（SCRNA-SEQ）由单个细胞中的基因表达组成。

A scRNA-seq workflow begins by isolating single cells from a piece of tissue either by microfluidic systems, laser capture microdissection, or fluorescence activated cell sorting (FACS), the last being the most commonly used technique. This isolated material becomes the starting point for RNA-seq analysis consisting of RNA isolation and mRNA enrichment, followed by fragmentation, conversion into complementary DNA molecule (cDNA), and the addition of sequencing adaptors. The cDNAs combined with the adaptors form the “DNA library” which will be sequenced and analyzed. Since the introduction of the method in 2009²，开发用于SCRNA-Seq的不同平台为研究人员提供了选择，以根据细胞隔离方法，吞吐量级别，覆盖率和灵敏度找到特定项目的最佳解决方案³。

2。patch-seq

神经元膜电位是可激发神经细胞的基本特征，可以用斑块钳技术解决。贴片钳包括使用与细胞膜紧密接触的微孔测量细胞的电性能⁴。RNA-seq的技术进步引起了许多包括斑块序列在内的大脑特异性RNA-seq方法。它提供了全细胞斑块钳记录的组合以及对同一孤立神经元的RNA-seq分析。遵循经典电生理学记录⁵，，，，cellular RNAs are extracted and sequenced. Initially established in 2015⁵，，，，the last five years have already seen innovations such as the implementation of more precise and differentiated RNA-seq measurements involving not only cytosolic RNAs but also distal RNAs localized at dendrites and axons⁶。斑块序列可以应用于不同的大脑区域，并对单个神经元的不同部位的整个转录组具有非常准确的视野。虽然斑块序列仍处于起步阶段，但这些功能使该方法非常有希望地研究与远端RNA和RNA贩运有关的疾病。

3。荧光in situRNA测序（Fisseq）

蛋白质表达的时空调节对于正常的脑功能和发育至关重要。在调节蛋白质亚细胞定位的途径中，mRNA定位是确保神经元细胞中正确蛋白质分布的主要机制⁷。Investigating RNA trafficking along neurons is crucial for understanding brain functionality. Initially, this question has been approached usingin situRNA分析。这项技术的最大限制是，它仅限于少数批准的生物标志物。2015年，Wyss Institute的George Church团队通过开发Fisseq（RNA的原位测序）方法来克服这一问题⁸。FISSEQ sequencesin situthe whole transcriptome of a fixed cell or tissue and links expression levels with spatial localization of the given molecule. The technique consists of a reverse transcription (RT) reaction on the fixed sample, which converts RNA into a complementary DNA (cDNA). The cDNA molecule is read before an amplification reaction occurs at the level of the molecule, incorporating a fluorescent signal that can be analyzed by confocal microscopy. The FISSEQ protocol is suitable for a large range of samples, including cultured cells, formalin fixed paraffin embedded (FFPE) and fresh frozen tissue samples, whole-mount果蝇embryos, and organoids⁸。

4。Multiplexed analysis of projections by sequencing (MAPSEQ)

神经回路是通过突触互连的神经元组，这些神经元可以位于远端大脑区域，但在功能上相互联系。追踪神经回路连通性的经典方法依赖于可以通过显微镜检测的染料注入神经元。最近，安东尼·扎多尔（Anthony Zador）的团队开发了一种新方法来跟踪单个神经元电路⁹。通过测序（MAPSEQ）对投影的多路复用分析包括将30bp序列的RNA条形码注入目标大脑区域。每个条形码将结合一个分子，该分子将通过单个神经元轴突途径传播到其最终突触目的地。之后，将大脑解剖为切片，并提取条形码的mRNA分子并进行测序，从源区域提供有关每个条形码RNA的电路模式的信息。这种新方法大大减少了单个神经元投射分析的时间，并大大增加了可以立即分析的神经元数量。

5.通过测序解决的条形码解剖结构（BARSEQ）

In 2019, Zador’s team introduced the upgraded version of MAPseq – barcoded anatomy resolved by sequencing (BARseq)¹⁰。与其祖先类似，Barseq涉及使用靶向RNA的条形码的高通量映射。这里的新颖性是进行测序in situ与MapSeq相比，将大脑切成切片，然后将RNA切成切片，然后将其萃取并测序。与使用MAPSEQ相比，此修改为条形码的初始位置以及跟踪电路提供了更高的空间分辨率。Zador和他的团队继续致力于下一代基于RNASEQ的映射技术，这将使我们更接近了解健康和神经病理学中的大脑连接性。

Conclusion

RNA-seq方法中的革命涉及许多研究领域中看到的趋势 - 重点是高通量，单细胞技术。我们可以看到，RNA-Seq技术也正在以更有针对性的方式发展，以寻找解决当今神经科学中最突出的局限性的解决方案。当涉及到大脑时，新型技术正在尝试将RNA-Seq与其他参数结合起来，而对于神经元功能至关重要，例如电活动，信使分子定位或神经元电路组织。

With the rapid progress in the development of these methods, scientists are expecting to gain better understanding in the complex structure, processes, and communications of the brain and hoping it will bring new therapeutic solutions in the years to follow.

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