我们感谢 Tuschl 博士, RNA 分子生物学实验室的主任, 以及他的实验室成员的支持和分享在他的实验室开发的技术, 并提供进入 RNAworld 管道。我们还感谢马库斯. Hafner 博士分享他的协议, 并详细描述了他的最初程序中使用的所有生化和酶步骤。

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作者希望披露一份载有本手稿中的一些数据的出版物是在《国际分子科学杂志》上发表的, Loudig et 。21。

本文提出了一种高重现性和健壮性的 cDNA 文库制备方法, 用于在 FFPE RNA 标本中存档的小 rna, 该协议是 Hafner et . 所描述的程序的修改和优化版本。22
本协议的所有步骤都已经过优化, 可用于从 FFPE 标本中恢复的较旧的存档和已损坏的总 RNA。该协议的关键步骤, 用于处理少量的 FFPE rna, 驻留在与18唯一3条形码适配器单独结扎后的所有 rna 样本 (即, 18 ?...

miRNAs 在福尔马林固定石蜡嵌入 (FFPE) 标本中保存得非常好,1,2,3。以往的研究表明, 这些短的监管非编码单链 rna 分子的表达可以成功地评估使用总 RNA 从 FFPE 样本, 并提供相关的基因表达数据时, 与原始新鲜组织4,5,6,7,8。当与大尺寸信使 rna 相比, 这已被证明是严重影响的 FFPE 组织处理 (甲醛, 热, 脱水,等), 内源性 RNases, 和年龄的标本, 小规模的 miRNAs (~ 18–24核苷酸) 似乎使它们耐降解和弹性的长期储存, 也证明通过 miRNA 表达研究, 优于高通量 mRNA 研究的存档标本9。miRNA 表达研究使用已存档的临床标本, 主要是在小规模分析中进行的, 证明了单一或多路定量 PCR 检测, 不同类型的微阵列技术, 最近的用于评估保存的 miRNAs 在这些化验的优化后的表达式10,11,12,13,14。
鉴于 miRNA 表达的失调与各种人类恶性肿瘤的发展有关, 而且有可能大量提供临床上标注的存档标本, 这已经变得明显, 这些小 RNA分子代表了潜在的癌症标志物的一个有希望的来源15,16,17,18。使用高通量基因表达技术 (如 miRNA) 的优点是, 当与 PCR 和/或微阵列19的目标技术相比较时, 可以对所有的转录记录进行全局评估。由于这个原因, 一个优化的, 负担得起的和易于适用的基因 cDNA 文库的协议, 从较旧的存档标本中制备小 rna 的方法进行了优化, 以实现大规模的回顾性研究20。
我们以前建立了一个同时 rna/dna 提取协议, 以分离恢复的 rna 和 dna 从旧的存档标本, 我们发现优于当代商业套件21。利用该提取协议, 从存档的 FFPE 组织中获取总 RNA, 对临床标本中保存的 miRNAs 的基因文库进行了35年的优化制备。此外, 在最近发表的一项研究中, 我们从临床分类导管癌原位(DCIS) 标本中制备了 cDNA 文库, 我们确定了定量 PCR 验证的差异表达 miRNAs, 表明这种特定的 miRNA 表达的变化可能是可检测到 DCIS 病变的患者谁发展乳腺癌时相比, DCIS 病变的病人谁不发展乳腺癌。
考虑到制作小 RNA cDNA 文库的商业套件的成本, 其终止的可能性, 以及使用版权/专利保护的试剂, 无法优化, 我们决定调整以前出版的基于实验室和无试剂盒的 3 ' 条形码 cDNA 文库在 FFPE 标本中存档的小 rna 的制备方案, 允许同时分析18个样本22。该议定书提供了一个理想和稳健的分步程序, 具有视觉和技术评估检查点, 这对于适应 FFPE RNA 标本是至关重要的, 并且具有很强的应用潜力, 可用于其他来源的损害或困难使用 RNA 材料。通过使用荧光 (例如、SYBR 金) 可检测到大型聚丙烯酰胺凝胶上结扎库的放射性标记尺寸标记, 改进了原始协议的适用性。这一优化的协议依赖于 3 ' 条形码适配器结扎18个单独的 FFPE RNA 标本, 然后汇集在一起进行 5 ' 适配器结扎, 反向转录, 和一个先导 PCR 分析, 以量身定制放大最终 cDNA图书馆在大规模的 PCR 放大, 纯化, 并在高通量排序器。

<table><tbody><tr><td>1% Triton x-100</td><td>Invitrogen</td><td>HFH10</td><td></td></tr><tr><td>10mM ATP</td><td>Ambion</td><td>AM8110G</td><td></td></tr><tr><td>10X dNTPs</td><td>Ambion</td><td>AM8110G</td><td></td></tr><tr><td>10x TBE</td><td>Thermofisher Scientific</td><td>15581044</td><td></td></tr><tr><td>14M Mercaptoethanol</td><td>Sigma</td><td>O3445I-100</td><td></td></tr><tr><td>20 nt ladder</td><td>Jena Bioscience</td><td>M-232S</td><td></td></tr><tr><td>20mg/ml Bovine Serum Albumine</td><td>Sigma</td><td>B8894-5ML</td><td></td></tr><tr><td>50X Titanium Taq</td><td>Clontech Laboratories</td><td>639208</td><td></td></tr><tr><td>Ammonium Persulfate</td><td>Fisher Scientific</td><td>7727-54-0</td><td></td></tr><tr><td>BRL Vertical Gel Electrophoresis System with glass plates and combs</td><td>GIBCO</td><td>V16</td><td></td></tr><tr><td>Dimethyl sulfoxide (DMSO)</td><td>Sigma</td><td>D9170-5VL</td><td></td></tr><tr><td>Eppendorf microcentrifuge 5424R</td><td>USA scientific</td><td>4054-4537Q</td><td></td></tr><tr><td>Eppndorf Thermomixer</td><td>USA scientific</td><td>4053-8223Q</td><td></td></tr><tr><td>Fisherbrand&amp;trade; Siliconized Low-Retention Microcentrifuge Tubes 1.5ml</td><td>Fisher Scientific</td><td>02-681-320</td><td></td></tr><tr><td>Gel Breaker Tube 0.5 ml</td><td>IST Engineering Inc,</td><td>3388-100</td><td></td></tr><tr><td>Gel electrophoresis apparatus 7cm x10cm- Mini-sub Cell GT with gel trays and combs</td><td>Biorad</td><td>1704446</td><td></td></tr><tr><td>Glycoblue</td><td>Ambion</td><td>AM9516</td><td></td></tr><tr><td>Jersey-Cote</td><td>LabScientific, Inc</td><td>1188</td><td></td></tr><tr><td>KcL 2M</td><td>Ambion</td><td>AM9640G</td><td></td></tr><tr><td>MgCl2 1M</td><td>Ambion</td><td>AM9530G</td><td></td></tr><tr><td>Minifuge dual rotor personal centrifuge</td><td>USA scientific</td><td>2641-0016</td><td></td></tr><tr><td>Model V16 polyacrylamide gel electrophoresis apparatus, glasses, combs, and spacers</td><td>Ciore Life Science</td><td>21070010</td><td></td></tr><tr><td>Oligonucleotides</td><td>IDT</td><td>Defined during order</td><td></td></tr><tr><td>Owl EasyCast B2 mini electrophoresis system- with gel trays and combs</td><td>Thermofisher Scientific</td><td>B2</td><td></td></tr><tr><td>Qiaquick Gel Extraction kit</td><td>Qiagen</td><td>28704</td><td></td></tr><tr><td>Restriction enzyme PmeI</td><td>NEB</td><td>R0560S</td><td></td></tr><tr><td>RNase-free water</td><td>Ambion</td><td>AM9932</td><td></td></tr><tr><td>Safe Imager 2.0</td><td>Life Technologies</td><td>G6600</td><td></td></tr><tr><td>Safe Imager 2.0 blue light transilluminator</td><td>Thermofisher</td><td>G6600</td><td></td></tr><tr><td>SeaKem LE agarose</td><td>Lonza</td><td>50002</td><td></td></tr><tr><td>Superscript III reverse transcription kit</td><td>Invitrogen</td><td>18080-044</td><td></td></tr><tr><td>SybrGold</td><td>Life Technologies</td><td>S11494</td><td></td></tr><tr><td>T4 RNA Ligase 1</td><td>NEB</td><td>M0204S</td><td></td></tr><tr><td>T4 RNA Ligase 2 Truncated K227Q</td><td>NEB</td><td>0351L</td><td></td></tr><tr><td>TEMED</td><td>Fisher Scientific</td><td>O3446I-100</td><td></td></tr><tr><td>Themocycler with heated lid</td><td>Applied Biosystem</td><td>4359659</td><td></td></tr><tr><td>Tris 1M pH 7.5</td><td>Invitrogen</td><td>15567027</td><td></td></tr><tr><td>Tris 1M pH8.0</td><td>Ambion</td><td>AM9855G</td><td></td></tr><tr><td>UltraPure Sequagel system concentrate, diluent, and buffer</td><td>National Diagnostics</td><td>EC-833</td><td></td></tr></tbody></table>

retrospective microrna sequencing: complementary dna library preparation protocol using formalin-fixed paraffin-embedded rna specimens

–Archived, 临床分类的福尔马林固定石蜡 (FFPE) 组织可以提供核酸的回顾性分子研究癌症的发展。通过使用晚期侵袭性疾病患者的非侵袭性或前恶性病变, 基因表达分析可以帮助识别易患癌症风险的早期分子改变。已经很好的描述, 从 FFPE 组织恢复的核酸发生了严重的物理损伤和化学修饰, 这使得他们的分析困难, 一般需要适应的化验。MicroRNAs (miRNAs), 但是, 它代表了一个小类 RNA 分子跨越只有18–24核苷酸, 已被证明经得起长期储存, 并已成功地分析了 FFPE 样品。在这里, 我们提出了一个 3 ' 条形码互补 DNA (cDNA) 库准备协议, 专门为分析从存档组织提取的小 rna, 最近证明是健壮的和高度重复使用存档临床标本贮存35年。此库准备很好地适应了被破坏/退化材料的多重分析, 其中 RNA 样品 (最多 18) 被结扎与单独 3 ' 条形码适配器, 然后汇集在一起为随后酵素和生化准备在分析之前。所有纯化都是由聚丙烯酰胺凝胶电泳 (页), 它允许大小特定的选择和充实的条形码小 RNA 品种。这种 cDNA 文库的制备很好地适应了微小的 RNA 输入, 作为一个先导聚合酶链反应 (PCR) 允许确定一个特定的放大周期, 以产生最佳数量的材料, 为下一代测序 ()。这一方法的优化, 以使用退化的 FFPE RNA 的标本归档了长达35年, 并提供了高度重现性的数据。

如本方法所述, 共有18个单独的 FFPE RNA 样本 (每100个) 被设置在单独的管中, 接受 3 ' adenylated 条形码寡核苷酸 T4 结扎过夜。第二天, 酶反应是热停用, 组合, 并沉淀在一个单一的管。rna 颗粒是悬浮和结扎 rna 分子分离在15% 变性聚丙烯酰胺凝胶 (页), 其中 RNA 寡核苷酸大小标记迁移在相邻的水井的页面凝胶, 用于选择适当大小的 3 ' 条形码小 rna (图 2)。切除...

Watch this Scientific Journal Video about Retrospective MicroRNA Sequencing: Complementary DNA Library Preparation Protocol Using Formalin-fixed Paraffin-embedded RNA Specimens at JoVE.com

Retrospective MicroRNA Sequencing: Complementary DNA Library Preparation Protocol Using Formalin-fixed Paraffin-embedded RNA Specimens

福尔马林固定石蜡镶嵌标本是人类疾病分子生物标志物的宝贵来源。在这里, 我们提出了一个基于实验室的 cDNA 文库准备协议, 最初是用新鲜的冷冻 RNA 设计的, 并对35年内储存的 microRNAs 进行了优化分析。

回顾性 MicroRNA 排序: 用福尔马林固定石蜡嵌入 RNA 标本补充 DNA 文库制备协议

&#8211;Archived, clinically classified formalin-fixed paraffin-embedded (FFPE) tissues can provide nucleic acids for retrospective molecular studies of ...

retrospective-microrna-sequencing-complementary-dna-library

Universidad de Cartagena UDEC

Research

JoVE Journal

Genetics

9.3K 次观看.  Hackensack University Medical Center. 福尔马林固定石蜡镶嵌标本是人类疾病分子生物标志物的宝贵来源。在这里, 我们提出了一个基于实验室的 cDNA 文库准备协议, 最初是用新鲜的冷冻 RNA 设计的, 并对35年内储存的 microRNAs 进行了优化分析。

视频: Retrospective MicroRNA Sequencing: Complementary DNA Library Preparation Protocol Using Formalin-fixed Paraffin-embedded RNA Specimens

Isolating, Sequencing and Analyzing Extracellular MicroRNAs from Human Mesenchymal Stem Cells

Extracellular and circulating RNAs (exRNA) are produced by many cell types of the body and exist in numerous bodily fluids such as saliva, plasma, serum, milk and urine. One subset of these RNAs are the posttranscriptional regulators &#8211; microRNAs (miRNAs). To delineate the miRNAs produced by specific cell types, in vitro culture systems can be used to harvest and profile exRNAs derived from one subset of cells. The secreted factors of mesenchymal stem cells are implicated in alleviating numerous diseases and is used as the in vitro model system here. This paper describes the process of collection, purification of small RNA and library generation to sequence extracellular miRNAs. ExRNAs from culture media differ from cellular RNA by being low RNA input samples, which calls for optimized procedures. This protocol provides a comprehensive guide to small exRNA sequencing from culture media, showing quality control checkpoints at each step during exRNA purification and sequencing.

This protocol demonstrates how to purify extracellular microRNAs from cell culture media for small RNA library construction and next generation sequencing. Various quality control checkpoints are described to allow readers to understand what to expect when working with low input samples like exRNAs.

人骨髓间充质干细胞细胞外微 rna 的分离、测序和分析

Extracellular and circulating RNAs (exRNA) are produced by many cell types of the body and exist in numerous bodily fluids such as saliva, plasma, serum, ...

科研

遗传学

A Complete Pipeline for Isolating and Sequencing MicroRNAs, and Analyzing Them Using Open Source Tools

Half of all human transcripts are thought to be regulated by microRNAs. Therefore, quantifying microRNA expression can reveal underlying mechanisms in disease states and provide therapeutic targets and biomarkers. Here, we detail how to accurately quantify microRNAs. Briefly, this method describes isolating microRNAs, ligating them to adaptors suitable for high-throughput sequencing, amplifying the final products, and preparing a sample library. Then, we explain how to align the obtained sequencing reads to microRNA hairpins, and quantify, normalize, and calculate their differential expression. Versatile and robust, this combined experimental workflow and bioinformatic analysis enables users to begin with tissue extraction and finish with microRNA quantification.

Here, we describe a step-by-step strategy for isolating small RNAs, enriching for microRNAs, and preparing samples for high-throughput sequencing. We then describe how to process sequence reads and align them to microRNAs, using open source tools.

用于隔离和测序微RNA并使用开源工具分析微RNA的完整管道

Half of all human transcripts are thought to be regulated by microRNAs. Therefore, quantifying microRNA expression can reveal underlying mechanisms in ...

Identification of Circular RNAs using RNA Sequencing

Circular RNAs (circRNAs) are a class of non-coding RNAs involved in functions including micro-RNA (miRNA) regulation, mediation of protein-protein interactions, and regulation of parental gene transcription. In classical next generation RNA sequencing (RNA-seq), circRNAs are typically overlooked as a result of poly-A selection during construction of mRNA libraries, or are found at very low abundance, and are therefore difficult to isolate and detect. Here, a circRNA library construction protocol was optimized by comparing library preparation kits, pre-treatment options and various total RNA input amounts. Two commercially available whole transcriptome library preparation kits, with and without RNase R pre-treatment, and using variable amounts of total RNA input (1 to 4 &#181;g), were tested. Lastly, multiple tissue types; including liver, lung, lymph node, and pancreas; as well as multiple brain regions; including the cerebellum, inferior parietal lobe, middle temporal gyrus, occipital cortex, and superior frontal gyrus; were compared to evaluate circRNA abundance across tissue types. Analysis of the generated RNA-seq data using six different circRNA detection tools (find_circ, CIRI, Mapsplice, KNIFE, DCC, and CIRCexplorer) revealed that a stranded total RNA library preparation kit with RNase R pre-treatment and 4 &#181;g RNA input is the optimal method for identifying the highest relative number of circRNAs. Consistent with previous findings, the highest enrichment of circRNAs was observed in brain tissues compared to other tissue types.

Circular RNAs (circRNAs) are non-coding RNAs that may have roles in transcriptional regulation and mediating interactions between proteins. Following assessment of different parameters for construction of circRNA sequencing libraries, a protocol was compiled utilizing stranded total RNA library preparation with RNase R pre-treatment and is presented here.

使用RNA测序识别循环RNA

Circular RNAs (circRNAs) are a class of non-coding RNAs involved in functions including micro-RNA (miRNA) regulation, mediation of protein-protein ...

使用 AQRNA-seq 分析 tRNA 动力学

AQRNA-seq for Quantifying Small RNAs

AQRNA-seq provides a direct linear relationship between sequencing read counts and small RNA copy numbers in a biological sample, thus enabling accurate quantification of the pool of small RNAs. The AQRNA-seq library preparation procedure described here involves the use of custom-designed sequencing linkers and a step for reducing methylation RNA modifications that block reverse transcription processivity, which results in an increased yield of full-length cDNAs. In addition, a detailed implementation of the accompanying bioinformatics pipeline is presented. This demonstration of AQRNA-seq was conducted through a quantitative analysis of the 45 tRNAs in Mycobacterium bovis BCG harvested on 5 selected days across a 20-day time course of nutrient deprivation and 6 days of resuscitation. Ongoing efforts to improve the efficiency and rigor of AQRNA-seq will also be discussed here. This includes exploring methods to obviate gel purification for mitigating primer dimer issues after PCR amplification and to increase the proportion of full-length reads to enable more accurate read mapping. Future enhancements to AQRNA-seq will be focused on facilitating automation and high-throughput implementation of this technology for quantifying all small RNA species in cell and tissue samples from diverse organisms.

作者聚焦:AQRNA-seq 在绘制小 RNA 和解开蛋白质翻译机制中的作用

Absolute quantification RNA sequencing (AQRNA-seq) is a technology developed to quantify the landscape of all small RNAs in biological mixtures. Here, both the library preparation and data processing steps of AQRNA-seq are demonstrated, quantifying changes in the transfer RNA (tRNA) pool in Mycobacterium bovis BCG during starvation-induced dormancy.

用于定量小 RNA 的 AQRNA-seq

AQRNA-seq provides a direct linear relationship between sequencing read counts and small RNA copy numbers in a biological sample, thus enabling accurate ...

Preparation of Small RNA Libraries for Sequencing from Early Mouse Embryos

MicroRNAs (miRNAs) are important for the complex regulation of cell fate decisions and developmental timing. In vivo studies of the contribution of miRNAs during early development are technically challenging due to the limiting cell number. Moreover, many approaches require a miRNA of interest to be defined in assays such as northern blotting, microarray, and qPCR. Therefore, the expression of many miRNAs and their isoforms have not been studied during early development. Here, we demonstrate a protocol for small RNA sequencing of sorted cells from early mouse embryos to enable relatively unbiased profiling of miRNAs in early populations of neural crest cells. We overcome the challenges of low cell input and size selection during library preparation using amplification and gel-based purification. We identify embryonic age as a variable accounting for variation between replicates and stage-matched mouse embryos must be used to accurately profile miRNAs in biological replicates. Our results suggest that this method can be broadly applied to profile the expression of miRNAs from other lineages of cells. In summary, this protocol can be used to study how miRNAs regulate developmental programs in different cell lineages of the early mouse embryo.

We describe a technique for profiling microRNAs in early mouse embryos. This protocol overcomes the challenge of low cell input and small RNA enrichment. This assay can be used to analyze changes in miRNA expression over time in different cell lineages of the early mouse embryo.

为早期小鼠胚胎测序准备小型RNA库

MicroRNAs (miRNAs) are important for the complex regulation of cell fate decisions and developmental timing. In vivo studies of the contribution of miRNAs ...

发育生物学

Collection and Processing of Lymph Nodes from Large Animals for RNA Analysis: Preparing for Lymph Node Transcriptomic Studies of Large Animal Species

Large animals (both livestock and wildlife) serve as important reservoirs of zoonotic pathogens, including Brucella, Mycobacterium bovis, Salmonella, and E. coli, and are useful for the study of pathogenesis and/or spread of the bacteria in natural hosts. With the key function of lymph nodes in the host immune response, lymph node tissues serve as a potential source of RNA for downstream transcriptomic analyses, in order to assess the temporal changes in gene expression in cells over the course of an infection. This article presents an overview of the process of lymph node collection, tissue sampling, and downstream RNA processing in livestock, using cattle (Bos taurus) as a model, with additional examples provided from the American bison (Bison bison). The protocol includes information about the location, identification, and removal of lymph nodes from multiple key sites in the body. Additionally, a biopsy sampling methodology is presented that allows for a consistency of sampling across multiple animals. Several considerations for sample preservation are discussed, including the generation of RNA suitable for downstream methodologies like RNA-sequencing and RT-PCR. Due to the long delays inherent in large animal vs. mouse time course studies, representative results from bison and bovine lymph node tissues are presented to describe the time course of the degradation in this tissue type, in the context of a review of previous methodological work on RNA degradation in other tissues. Overall, this protocol will be useful to both veterinary researchers beginning transcriptome projects on large animal samples and to molecular biologists interested in learning techniques for in vivo tissue sampling and in vitro processing.

This protocol provides an overview of procedures for the isolation of RNA for the transcriptomic profiling of lymph node tissues from large animals, including steps in the identification and excision of lymph nodes from livestock and wildlife, sampling approaches to provide consistency across multiple animals, and considerations plus representative results for the post-collection preservation and processing for RNA analysis.

大动物淋巴结的采集与处理 RNA 分析: 大动物 Transcriptomic 淋巴结的制备研究

Large animals (both livestock and wildlife) serve as important reservoirs of zoonotic pathogens, including Brucella, Mycobacterium bovis, Salmonella, and ...

免疫与感染

Laser Capture Microdissection of Glioma Subregions for Spatial and Molecular Characterization of Intratumoral Heterogeneity, Oncostreams, and Invasion

Gliomas are primary brain tumors characterized by their invasiveness and heterogeneity. Specific histological patterns such as pseudopalisades, microvascular proliferation, mesenchymal transformation and necrosis characterize the histological heterogeneity of high-grade gliomas. Our laboratory has demonstrated that the presence of high densities of mesenchymal cells, named oncostreams, correlate with tumor malignancy. We have developed a unique approach to understand the mechanisms that underlie glioma&#39;s growth and invasion. Here, we describe a comprehensive protocol that utilizes laser capture microdissection (LMD) and RNA sequencing to analyze differential mRNA expression of intra-tumoral heterogeneous multicellular structures (i.e., mesenchymal areas or areas of tumor invasion). This method maintains good tissue histology and RNA integrity. Perfusion, freezing, embedding, sectioning, and staining were optimized to preserve morphology and obtain high-quality laser microdissection samples. The results indicate that perfusion of glioma bearing mice using 30% sucrose provides good morphology and RNA quality. In addition, staining tumor sections with 4% Cresyl violet and 0.5% eosin results in good nuclear and cellular staining, while preserving RNA integrity. The method described is sensitive and highly reproducible and it can be utilized to study tumor morphology in various tumor models. In summary, we describe a complete method to perform LMD that preserves morphology and RNA quality for sequencing to study the molecular features of heterogeneous multicellular structures within solid tumors.

Laser microdissection (LMD) is a sensitive and highly reproducible technique that can be used to uncover pathways that mediate glioma heterogeneity and invasion. Here, we describe an optimized protocol to isolate discrete areas from glioma tissue using laser LMD followed by transcriptomic analysis.

激光捕获胶质瘤分区域的微切，用于肿瘤内异质性、肿瘤流和入侵的空间和分子表征

Gliomas are primary brain tumors characterized by their invasiveness and heterogeneity. Specific histological patterns such as pseudopalisades, ...

神经科学

Highly Efficient Ligation of Small RNA Molecules for MicroRNA Quantitation by High-Throughput Sequencing

MiRNA cloning and high-throughput sequencing, termed miR-Seq, stands alone as a transcriptome-wide approach to quantify miRNAs with single nucleotide resolution. This technique captures miRNAs by attaching 3&#8217; and 5&#8217; oligonucleotide adapters to miRNA molecules and allows de novo miRNA discovery. Coupling with powerful next-generation sequencing platforms, miR-Seq has been instrumental in the study of miRNA biology. However, significant biases introduced by oligonucleotide ligation steps have prevented miR-Seq from being employed as an accurate quantitation tool. Previous studies demonstrate that biases in current miR-Seq methods often lead to inaccurate miRNA quantification with errors up to 1,000-fold for some miRNAs1,2. To resolve these biases imparted by RNA ligation, we have developed a small RNA ligation method that results in ligation efficiencies of over 95% for both 3&#8217; and 5&#8242; ligation steps. Benchmarking this improved library construction method using equimolar or differentially mixed synthetic miRNAs, consistently yields reads numbers with less than two-fold deviation from the expected value. Furthermore, this high-efficiency miR-Seq method permits accurate genome-wide miRNA profiling from in vivo total RNA samples2.

MicroRNAs (miRNAs) are a widely conserved class of regulatory molecules. Here we describe a miRNA cloning method that relies upon two potent ligation steps followed by high-throughput sequencing. Our method permits accurate genome-wide quantitation of miRNAs.

小RNA分子的高效结扎的微小RNA定量高通量测序

MiRNA cloning and high-throughput sequencing, termed miR-Seq, stands alone as a transcriptome-wide approach to quantify miRNAs with single nucleotide ...

回顾性 MicroRNA 排序: 用福尔马林固定石蜡嵌入 RNA 标本补充 DNA 文库制备协议

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此视频中的章节

人骨髓间充质干细胞细胞外微 rna 的分离、测序和分析

用于隔离和测序微RNA并使用开源工具分析微RNA的完整管道

使用RNA测序识别循环RNA

用于定量小 RNA 的 AQRNA-seq

用于定量小 RNA 的 AQRNA-seq

用于定量小 RNA 的 AQRNA-seq

为早期小鼠胚胎测序准备小型RNA库

大动物淋巴结的采集与处理 RNA 分析: 大动物 Transcriptomic 淋巴结的制备研究

激光捕获胶质瘤分区域的微切，用于肿瘤内异质性、肿瘤流和入侵的空间和分子表征

小RNA分子的高效结扎的微小RNA定量高通量测序