Biology Methods and Protocols最新文献

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Preparation and processing of dried blood spots for microRNA sequencing. 用于微小RNA测序的干血斑的制备和处理。

IF 3.6 Q3 BIOCHEMICAL RESEARCH METHODS

Biology Methods and Protocols

Pub Date : 2023-09-20 eCollection Date: 2023-01-01 DOI: 10.1093/biomethods/bpad020

Alice Morgunova, Pascal Ibrahim, Gary Gang Chen, Saché M Coury, Gustavo Turecki, Michael J Meaney, Anthony Gifuni, Ian H Gotlib, Corina Nagy, Tiffany C Ho, Cecilia Flores

Dried blood spots (DBS) are biological samples commonly collected from newborns and in geographic areas distanced from laboratory settings for the purposes of disease testing and identification. MicroRNAs (miRNAs)-small non-coding RNAs that regulate gene activity at the post-transcriptional level-are emerging as critical markers and mediators of disease, including cancer, infectious diseases, and mental disorders. This protocol describes optimized procedural steps for utilizing DBS as a reliable source of biological material for obtaining peripheral miRNA expression profiles. We outline key practices, such as the method of DBS rehydration that maximizes RNA extraction yield, and the use of degenerate oligonucleotide adapters to mitigate ligase-dependent biases that are associated with small RNA sequencing. The standardization of miRNA readout from DBS offers numerous benefits: cost-effectiveness in sample collection and processing, enhanced reliability and consistency of miRNA profiling, and minimal invasiveness that facilitates repeated testing and retention of participants. The use of DBS-based miRNA sequencing is a promising method to investigate disease mechanisms and to advance personalized medicine.

干血点（DBS）是通常从新生儿身上采集的生物样本，位于远离实验室的地理区域，用于疾病检测和鉴定。微小RNA（miRNA）-在转录后水平调节基因活性的小型非编码RNA正在成为疾病的关键标志物和介质，包括癌症、传染病和精神疾病。该方案描述了利用DBS作为获得外周miRNA表达谱的可靠生物材料来源的优化程序步骤。我们概述了关键的实践，如最大限度地提高RNA提取产量的DBS再水合方法，以及使用简并寡核苷酸适配器来减轻与小RNA测序相关的连接酶依赖性偏差。DBS中miRNA读数的标准化提供了许多好处：样本收集和处理的成本效益，提高了miRNA图谱的可靠性和一致性，以及促进重复测试和保留参与者的最小侵袭性。使用基于DBS的miRNA测序是研究疾病机制和推进个性化医学的一种很有前途的方法。

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引用次数: 0

An advanced protocol for profiling RNA-binding proteins in Arabidopsis using plant phase extraction 一种利用植物相萃取分析拟南芥rna结合蛋白的先进方案

IF 3.6 Q3 BIOCHEMICAL RESEARCH METHODS

Biology Methods and Protocols

Pub Date : 2023-08-10 DOI: 10.1093/biomethods/bpad016

Yong Zhang, Ye Xu, T. Skaggs, J. F. Ferreira, Xuemei Chen, D. Sandhu

RNA-binding proteins (RBPs) are key players in regulating cell fate and essential developmental processes. Systematic profiling of the RNA-binding proteome (RBPome) is thus indispensable for researchers aiming to understand the mechanisms of post-transcriptional gene regulation. RBPome identification methods developed in humans, mice, and bacteria have successfully identified RBPomes in these organisms. However, the biochemical and genetic complexities of plant tissues have greatly hindered the effectiveness of these methods in plants. Moreover, plant RBPs have been predominantly discovered through oligo d(T) based affinity purification (RNA-interactome capture, RIC). Since polyadenylated RNA only accounts for less than 5% of the total RNA population in eukaryotic cells, there is a pressing need to develop a comprehensive yet unbiased method to capture the full spectrum of RBPs in plants. Here, we describe a detailed protocol of Plant Phase Extraction (PPE), a recently developed method to identify RBPs in Arabidopsis [1]. The PPE method enables the efficient enrichment of both poly(A) and non-poly(A) RBPs from various tissues quickly and reproducibly. Most importantly, PPE allows for unveiling dynamic RBP-RNA interactions under various abiotic and biotic stress conditions and during different plant developmental stages. This provides a much broader and more accurate understanding of plant RBPs, marking a significant advancement in plant molecular biology.

RNA结合蛋白（RBPs）是调节细胞命运和重要发育过程的关键参与者。因此，RNA结合蛋白质组（RBPome）的系统分析对于旨在了解转录后基因调控机制的研究人员来说是必不可少的。在人类、小鼠和细菌中开发的RBPome鉴定方法已成功鉴定出这些生物体中的RBPomes。然而，植物组织的生物化学和遗传复杂性极大地阻碍了这些方法在植物中的有效性。此外，植物RBPs主要是通过基于寡核苷酸（T）的亲和纯化（RNA相互作用组捕获，RIC）发现的。由于多腺苷酸化的RNA仅占真核细胞中总RNA种群的不到5%，因此迫切需要开发一种全面而无偏见的方法来捕获植物中的全谱RBPs。在这里，我们描述了植物相提取（PPE）的详细方案，这是一种最近开发的在拟南芥中鉴定RBPs的方法[1]。PPE方法能够快速且可重复地从各种组织中高效富集聚（A）和非聚（A”RBP。最重要的是，PPE可以揭示在各种非生物和生物胁迫条件下以及不同植物发育阶段的RBP-RNA动态相互作用。这为植物RBPs提供了更广泛、更准确的理解，标志着植物分子生物学的重大进步。

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引用次数: 0

Protocol for transcriptome assembly by the TransBorrow algorithm TransBorrow算法转录组组装协议

Q3 BIOCHEMICAL RESEARCH METHODS

Biology Methods and Protocols

Pub Date : 2023-01-01 DOI: 10.1093/biomethods/bpad028

Dengyi Zhao, Juntao Liu, Ting Yu

Abstract High-throughput RNA-seq enables comprehensive analysis of the transcriptome for various purposes. However, this technology generally generates massive amounts of sequencing reads with a shorter read length. Consequently, fast, accurate, and flexible tools are needed for assembling raw RNA-seq data into full-length transcripts and quantifying their expression levels. In this protocol, we report TransBorrow, a novel transcriptome assembly software specifically designed for short RNA-seq reads. TransBorrow is employed in conjunction with a splice-aware alignment tool (e.g. Hisat2 and Star) and some other transcriptome assembly tools (e.g. StringTie, Cufflinks, and Scallop). The protocol encompasses all necessary steps, starting from downloading and processing raw sequencing data to assembling the full-length transcripts and quantifying their expressed abundances. The execution time of the protocol may vary depending on the sizes of processed datasets and computational platforms.

高通量RNA-seq能够对各种目的的转录组进行全面分析。然而，这种技术通常产生大量的测序读取，读取长度较短。因此，需要快速、准确和灵活的工具来将原始RNA-seq数据组装成全长转录本并量化其表达水平。在本协议中，我们报告了TransBorrow，一种专门设计用于短RNA-seq读取的新型转录组组装软件。TransBorrow与拼接感知比对工具(例如Hisat2和Star)和其他转录组组装工具(例如StringTie, Cufflinks和扇贝)结合使用。该方案包括所有必要的步骤，从下载和处理原始测序数据到组装全长转录本并量化其表达丰度。协议的执行时间可能根据处理的数据集和计算平台的大小而变化。

引用次数: 0

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