Name: retrospective microrna sequencing: complementary dna library preparation protocol using formalin-fixed paraffin-embedded rna specimens
Uploaded: 2018-05-05T13:00:00.000+00:00
Duration: 12 min 24 s
Description: Watch this Scientific Journal Video about Retrospective MicroRNA Sequencing: Complementary DNA Library Preparation Protocol Using Formalin-fixed Paraffin-embedded RNA Specimens at JoVE.com

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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Invest. 94, 350-358 (2014).</li><li>Loudig, O., Wan, T., Ye, K., Lin, J., Wang, Y., Ramnauth, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+and+Adaptation+of+a+Laboratory+Based+cDNA+Library+Preparation+Protocol+for+Retrospective+Sequencing+of+Archived+MicroRNAs+from+up+to+35-Year-Old+Clinical+FFPE+Specimens.">Evaluation and Adaptation of a Laboratory Based cDNA Library Preparation Protocol for Retrospective Sequencing of Archived MicroRNAs from up to 35-Year-Old Clinical FFPE Specimens.</a> Int J Mol Sci. 18, e627 (2017).</li><li>Kotorashvili, A., Ramnauth, A., Liu, C., Lin, J., Ye, K., Kim, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effective+DNA/RNA+co-extraction+for+analysis+of+microRNAs,+mRNAs,+and+genomic+DNA+from+formalin-fixed+paraffin-embedded+specimens.">Effective DNA/RNA co-extraction for analysis of microRNAs, mRNAs, and genomic DNA from formalin-fixed paraffin-embedded specimens.</a> PLoS One. 7 (4), e34683 (2012).</li><li>Hafner, M., Renwick, N., Farazi, T. 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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

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 ...

Sequencing Small Non-coding RNA from Formalin-fixed Tissues and Serum-derived Exosomes from Castration-resistant Prostate Cancer Patients

Ablation of androgen receptor (AR) signaling by androgen deprivation is the goal of the first line of therapy for prostate cancer that initially results in cancer regression. However, in a significant number of cases, the disease progresses to advanced, castration-resistant prostate cancer (CRPC), which has limited therapeutic options and is often aggressive. Distant metastasis is mostly observed at this stage of the aggressive disease. CRPC is treated by a second generation of AR pathway inhibitors that improve survival initially, followed by the emergence of therapy resistance. Neuroendocrine prostate cancer (NEPC) is a rare variant of prostate cancer (PCa) that often develops as a result of therapy resistance via a transdifferentiation process known as neuroendocrine differentiation (NED), wherein PCa cells undergo a lineage switch from adenocarcinomas and show increased expression of neuroendocrine (NE) lineage markers. In addition to the genomic alterations that drive the progression and transdifferentiation to NEPC, epigenetic factors and microenvironmental cues are considered essential players in driving disease progression. This manuscript provides a detailed protocol to identify the epigenetic drivers (i.e., small non-coding RNAs) that are associated with advanced PCa. Using purified microRNAs from formalin-fixed paraffin-embedded (FFPE) metastatic tissues and corresponding serum-derived extracellular vesicles (EVs), the protocol describes how to prepare libraries with appropriate quality control for sequencing microRNAs from these sample sources. Isolating RNA from both FFPE and EVs is often challenging because most of it is either degraded or is limited in quantity. This protocol will elaborate on different methods to optimize the RNA inputs and cDNA libraries to yield most specific reads and high-quality data upon sequencing.

Therapy resistance often develops in patients with advanced prostate cancer, and in some cases, cancer progresses to a lethal subtype called neuroendocrine prostate cancer. Assessing the small non-coding RNA-mediated molecular changes that facilitate this transition would allow better disease stratification and identification of causal mechanisms that lead to development of neuroendocrine prostate cancer.

从卡西甲抗性前列腺癌患者的甲酸固定组织和血清衍生的外源体对小型非编码RNA进行测序

Ablation of androgen receptor (AR) signaling by androgen deprivation is the goal of the first line of therapy for prostate cancer that initially results ...

癌症研究

Optimization for Sequencing and Analysis of Degraded FFPE-RNA Samples

Gene expression analysis by RNA sequencing (RNA-seq) enables unique insights into clinical samples that can potentially lead to mechanistic understanding of the basis of various diseases as well as resistance and/or susceptibility mechanisms. However, FFPE tissues, which represent the most common method for preserving tissue morphology in clinical specimens, are not the best sources for gene expression profiling analysis. The RNA obtained from such samples is often degraded, fragmented, and chemically modified, which leads to suboptimal sequencing libraries. In turn, these generate poor quality sequence data that may not be reliable for gene expression analysis and mutation discovery. In order to make the most of FFPE samples and obtain the best possible data from low quality samples, it is important to take certain precautions while planning experimental design, preparing sequencing libraries, and during data analysis. This includes the use of appropriate metrics for precise sample quality control (QC), identifying the best methods for various steps during the sequencing library generation, and careful library QC. In addition, applying correct software tools and parameters for sequence data analysis is critical in order to identify artifacts in RNA-seq data, filter out contamination and low quality reads, assess uniformity of gene coverage, and measure the reproducibility of gene expression profiles among biological replicates. These steps can ensure high accuracy and reproducibility for profiling of very heterogeneous RNA samples. Here we describe the various steps for sample QC, library preparation and QC, sequencing, and data analysis that can help to increase the amount of useful data obtained from low quality RNA, such as that obtained from FFPE-RNA tissues.

This method describes the steps to improve the quality and quantity of sequence data that can be obtained from formalin-fixed paraffin-embedded (FFPE) RNA samples. We describe the methodology to more accurately assess the quality of FFPE-RNA samples, prepare sequencing libraries, and analyze the data from FFPE-RNA samples.

降解FFPE-RNA样品测序与分析优化

Gene expression analysis by RNA sequencing (RNA-seq) enables unique insights into clinical samples that can potentially lead to mechanistic understanding ...

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

Single Read and Paired End mRNA-Seq Illumina Libraries from 10 Nanograms Total RNA

Whole transcriptome sequencing by mRNA-Seq is now used extensively to perform global gene expression, mutation, allele-specific expression and other genome-wide analyses. mRNA-Seq even opens the gate for gene expression analysis of non-sequenced genomes. mRNA-Seq offers high sensitivity, a large dynamic range and allows measurement of transcript copy numbers in a sample. Illumina&#x2019;s genome analyzer performs sequencing of a large number (&gt; 107) of relatively short sequence reads (&lt; 150 bp).The &quot;paired end&quot; approach, wherein a single long read is sequenced at both its ends, allows for tracking alternate splice junctions, insertions and deletions, and is useful for de novo transcriptome assembly.
One of the major challenges faced by researchers is a limited amount of starting material. For example, in experiments where cells are harvested by laser micro-dissection, available starting total RNA may measure in nanograms. Preparation of mRNA-Seq libraries from such samples have been described1, 2 but involves significant PCR amplification that may introduce bias. Other RNA-Seq library construction procedures with minimal PCR amplification have been published3, 4 but require microgram amounts of starting total RNA.
Here we describe a protocol for the Illumina Genome Analyzer II platform for mRNA-Seq sequencing for library preparation that avoids significant PCR amplification and requires only 10 nanograms of total RNA. While this protocol has been described previously and validated for single-end sequencing5, where it was shown to produce directional libraries without introducing significant amplification bias, here we validate it further for use as a paired end protocol. We selectively amplify polyadenylated messenger RNAs from starting total RNA using the T7 based Eberwine linear amplification method, coined &quot;T7LA&quot; (T7 linear amplification). The amplified poly-A mRNAs are fragmented, reverse transcribed and adapter ligated to produce the final sequencing library. For both single read and paired end runs, sequences are mapped to the human transcriptome6 and normalized so that data from multiple runs can be compared. We report the gene expression measurement in units of transcripts per million (TPM), which is a superior measure to RPKM when comparing samples7.

Here we describe a method for preparation of both single read and paired end Illumina mRNA-Seq sequencing libraries for gene expression analysis based on T7 linear RNA amplification. This protocol requires only 10 nanograms of starting total RNA and generates highly consistent libraries representing whole transcripts.

单次读取和配对的最终从10微克总RNA的mRNA - Seq的Illumina的图书馆

Whole transcriptome sequencing by mRNA-Seq is now used extensively to perform global gene expression, mutation, allele-specific expression and other ...

生物学

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 ...

Targeted RNA Sequencing Assay to Characterize Gene Expression and Genomic Alterations

RNA sequencing (RNAseq) is a versatile method that can be utilized to detect and characterize gene expression, mutations, gene fusions, and noncoding RNAs. Standard RNAseq requires 30 - 100 million sequencing reads and can include multiple RNA products such as mRNA and noncoding RNAs. We demonstrate how targeted RNAseq (capture) permits a focused study on selected RNA products using a desktop sequencer. RNAseq capture can characterize unannotated, low, or transiently expressed transcripts that may otherwise be missed using traditional RNAseq methods. Here we describe the extraction of RNA from cell lines, ribosomal RNA depletion, cDNA synthesis, preparation of barcoded libraries, hybridization and capture of targeted transcripts and multiplex sequencing on a desktop sequencer. We also outline the computational analysis pipeline, which includes quality control assessment, alignment, fusion detection, gene expression quantification and identification of single nucleotide variants. This assay allows for targeted transcript sequencing to characterize gene expression, gene fusions, and mutations.

We describe a targeted RNA sequencing-based method that includes preparation of indexed cDNA libraries, hybridization and capture with custom probes and data analysis to interrogate selected transcripts for gene expression, mutations, and gene fusions. Targeted RNAseq permits cost-effective, rapid evaluation of selected transcripts on a desktop sequencer.

有针对性的RNA测序分析表征基因表达和基因组改变

RNA sequencing (RNAseq) is a versatile method that can be utilized to detect and characterize gene expression, mutations, gene fusions, and noncoding ...

Preparation of Formalin-fixed Paraffin-embedded Tissue Cores for both RNA and DNA Extraction

Formalin-fixed paraffin embedded tissue (FFPET) represents a valuable, well-annotated substrate for molecular investigations. The utility of FFPET in molecular analysis is complicated both by heterogeneous tissue composition and low yields when extracting nucleic acids. A literature search revealed a paucity of protocols addressing these issues, and none that showed a validated method for simultaneous extraction of RNA and DNA from regions of interest in FFPET. This method addresses both issues. Tissue specificity was achieved by mapping cancer areas of interest on microscope slides and transferring annotations onto FFPET blocks. Tissue cores were harvested from areas of interest using 0.6 mm microarray punches. Nucleic acid extraction was performed using a commercial FFPET extraction system, with modifications to homogenization, deparaffinization, and Proteinase K digestion steps to improve tissue digestion and increase nucleic acid yields. The modified protocol yields sufficient quantity and quality of nucleic acids for use in a number of downstream analyses, including a multi-analyte gene expression platform, as well as reverse transcriptase coupled real time PCR analysis of mRNA expression, and methylation-specific PCR (MSP) analysis of DNA methylation.

This modified extraction protocol improves RNA and DNA yields from more precisely targeted regions of interest in histopathologic tissue blocks.

福尔马林固定石蜡包埋的组织核心的制备RNA和DNA提取

Formalin-fixed paraffin embedded tissue (FFPET) represents a valuable, well-annotated substrate for molecular investigations. The utility of FFPET in ...