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。

<ol><li>Peiró-Chova, L., Peña-Chilet, M., López-, J. A., García-Gimenez, J. L., Alonso-Yuste, E., Burgues, O., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((High+stability+of+microRNAs+in+tissue+samples+of+compromised+quality%5BTitle%5D)+AND+%22Virchows.+Arch%22%5BJournal%5D)">High stability of microRNAs in tissue samples of compromised quality.</a> Virchows. Arch. 463, 765-774 (2013).</li>
<li>Klopfleisch, R., Weiss, A. T., Gruber, A. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Excavation+of+a+buried+treasure--DNA%2C+mRNA%2C+miRNA+and+protein+analysis+in+formalin+fixed%2C+paraffin+embedded+tissues%5BTitle%5D)+AND+%22Histol.+Histopathol%22%5BJournal%5D)">Excavation of a buried treasure--DNA, mRNA, miRNA and protein analysis in formalin fixed, paraffin embedded tissues.</a> Histol. Histopathol. 26, 797-810 (2011).</li>
<li>Kakimoto, Y., Kamiguchi, H., Ochiai, E., Satoh, F., Osawa, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((MicroRNA+Stability+in+Postmortem+FFPE+Tissues%3A+Quantitative+Analysis+Using+Autoptic+Samples+from+Acute+Myocardial+Infarction+Patients%5BTitle%5D)+AND+%22PLoS+One%22%5BJournal%5D)">MicroRNA Stability in Postmortem FFPE Tissues: Quantitative Analysis Using Autoptic Samples from Acute Myocardial Infarction Patients.</a> PLoS One. 10 (6), e0129338(2015).</li>
<li>Giricz, O., Reynolds, P. A., Ramnauth, A., Liu, C., Wang, T., Stead, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Hsa-miR-375+is+differentially+expressed+during+breast+lobular+neoplasia+and+promotes+loss+of+mammary+acinar+polarity%5BTitle%5D)+AND+%22J.+Pathol%22%5BJournal%5D)">Hsa-miR-375 is differentially expressed during breast lobular neoplasia and promotes loss of mammary acinar polarity.</a> J. Pathol. 226, 108-119 (2012).</li>
<li>Hui, A. B., Shi, W., Boutros, P. C., Miller, N., Pintilie, M., Fyles, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Robust+global+micro-RNA+profiling+with+formalin-fixed+paraffin-embedded+breast+cancer+tissues%5BTitle%5D)+AND+%22Lab.+Invest%22%5BJournal%5D)">Robust global micro-RNA profiling with formalin-fixed paraffin-embedded breast cancer tissues.</a> Lab. Invest. 89, 597-606 (2009).</li>
<li>Zhang, X., Chen, J., Radcliffe, T., Lebrun, D. P., Tron, V. A., Feilotter, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((An+array-based+analysis+of+microRNA+expression+comparing+matched+frozen+and+formalin-fixed+paraffin-embedded+human+tissue+samples%5BTitle%5D)+AND+%22J.+Mol.+Diagn%22%5BJournal%5D)">An array-based analysis of microRNA expression comparing matched frozen and formalin-fixed paraffin-embedded human tissue samples.</a> J. Mol. Diagn. 10, 513-519 (2008).</li>
<li>Kolbert, C. P., Feddersen, R. M., Rakhshan, F., Grill, D. E., Simon, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Multi-platform+analysis+of+microRNA+expression+measurements+in+RNA+from+fresh+frozen+and+FFPE+tissues%5BTitle%5D)+AND+%22PLoS+One%22%5BJournal%5D)">Multi-platform analysis of microRNA expression measurements in RNA from fresh frozen and FFPE tissues.</a> PLoS One. 8, e52517(2013).</li>
<li>Meng, W., McElroy, J. P., Volinia, S., Palatini, J., Warner, S., Ayers, L. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Comparison+of+microRNA+deep+sequencing+of+matched+formalin-fixed+paraffin-embedded+and+fresh+frozen+cancer+tissues%5BTitle%5D)+AND+%22PLoS+One%22%5BJournal%5D)">Comparison of microRNA deep sequencing of matched formalin-fixed paraffin-embedded and fresh frozen cancer tissues.</a> PLoS One. 8 (5), e64393(2013).</li>
<li>Liu, A., Tetzlaff, M. T., Vanbelle, P., Elder, D., Feldman, M., Tobias, J. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((MicroRNA+expression+profiling+outperforms+mRNA+expression+profiling+in+formalin-fixed+paraffin-embedded+tissues%5BTitle%5D)+AND+%22Int.+J.+Clin.+Exp.+Pathol%22%5BJournal%5D)">MicroRNA expression profiling outperforms mRNA expression profiling in formalin-fixed paraffin-embedded tissues.</a> Int. J. Clin. Exp. Pathol. 2, 519-527 (2009).</li>
<li>Dijkstra, J. R., Mekenkamp, L. J., Teerenstra, S., De Krijger, I., Nagtegaal, I. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((MicroRNA+expression+in+formalin-fixed+paraffin+embedded+tissue+using+real+time+quantitative+PCR%3A+the+strengths+and+pitfalls%5BTitle%5D)+AND+%22J+Cell+Mol+Med%22%5BJournal%5D)">MicroRNA expression in formalin-fixed paraffin embedded tissue using real time quantitative PCR: the strengths and pitfalls.</a> J Cell Mol Med. 16, 683-690 (2012).</li>
<li>Siebolts, U., Varnholt, H., Drebber, U., Dienes, H. P., Wickenhauser, C., Odenthal, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Tissues+from+routine+pathology+archives+are+suitable+for+microRNA+analyses+by+quantitative+PCR%5BTitle%5D)+AND+%22J.+Clin.+Pathol%22%5BJournal%5D)">Tissues from routine pathology archives are suitable for microRNA analyses by quantitative PCR.</a> J. Clin. Pathol. 62, 84-88 (2009).</li>
<li>Jang, J. S., Simon, V. A., Feddersen, R. M., Rakhshan, F., Schultz, D. A., Zschunke, M. A., Lingle, W. L., Kolbert, C. P., Jen, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Quantitative+miRNA+expression+analysis+using+fluidigm+microfluidics+dynamic+arrays%5BTitle%5D)+AND+%22BMC+Genomics%22%5BJournal%5D)">Quantitative miRNA expression analysis using fluidigm microfluidics dynamic arrays.</a> BMC Genomics. 12, 144(2011).</li>
<li>Andreasen, D., Fog, J. U., Biggs, W., Salomon, J., Dahslveen, I. K., Baker, A., Mouritzen, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Improved+microRNA+quantification+in+total+RNA+from+clinical+samples%5BTitle%5D)+AND+%22Methods%22%5BJournal%5D)">Improved microRNA quantification in total RNA from clinical samples.</a> Methods. 50 (4), S6-S9 (2010).</li>
<li>Kelly, A. D., Hill, K. E., Correll, M., Wang, Y. E., Rubio, R., Duan, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Next-generation+sequencing+and+microarray-based+interrogation+of+microRNAs+from+formalin-fixed%2C+paraffin-embedded+tissue%3A+preliminary+assessment+of+cross-platform+concordance%5BTitle%5D)+AND+%22Genomics%22%5BJournal%5D)">Next-generation sequencing and microarray-based interrogation of microRNAs from formalin-fixed, paraffin-embedded tissue: preliminary assessment of cross-platform concordance.</a> Genomics. 102, 8-14 (2013).</li>
<li>Visone, R., Croce, C. M. <a target="_blank" href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2671346/">MiRNAs and cancer.</a> Am. J. Pathol. 174, 1131-1138 (2009).</li>
<li>Lu, J., Getz, G., Miska, E. A., Alvarez-Saavedra, E., Lamb, J., Peck, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((MicroRNA+expression+profiles+classify+human+cancers%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">MicroRNA expression profiles classify human cancers.</a> Nature. 435, 834-838 (2005).</li>
<li>Cho, W. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((MicroRNAs%3A+potential+biomarkers+for+cancer+diagnosis%2C+prognosis+and+targets+for+therapy%5BTitle%5D)+AND+%22Int.+J.+Biochem.+Cell.+Biol%22%5BJournal%5D)">MicroRNAs: potential biomarkers for cancer diagnosis, prognosis and targets for therapy.</a> Int. J. Biochem. Cell. Biol. 42, 1273-1281 (2010).</li>
<li>Hui, A., How, C., Ito, E., Liu, F. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Micro-RNAs+as+diagnostic+or+prognostic+markers+in+human+epithelial+malignancies%5BTitle%5D)+AND+%22BMC+Cancer%22%5BJournal%5D)">Micro-RNAs as diagnostic or prognostic markers in human epithelial malignancies.</a> BMC Cancer. 11, 500(2011).</li>
<li>Tam, S., de Borja, R., Tsao, M. S., McPherson, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Robust+global+microRNA+expression+profiling+using+next-generation+sequencing+technologies%5BTitle%5D)+AND+%22Lab.+Invest%22%5BJournal%5D)">Robust global microRNA expression profiling using next-generation sequencing technologies.</a> Lab. 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/pubmed?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%5BTitle%5D)+AND+%22Int+J+Mol+Sci%22%5BJournal%5D)">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/pubmed?term=((Effective+DNA%2FRNA+co-extraction+for+analysis+of+microRNAs%2C+mRNAs%2C+and+genomic+DNA+from+formalin-fixed+paraffin-embedded+specimens%5BTitle%5D)+AND+%22PLoS+One%22%5BJournal%5D)">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. A., Mihailović, A., Pena, J. T., Tuschl, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Barcoded+cDNA+library+preparation+for+small+RNA+profiling+by+next-generation+sequencing%5BTitle%5D)+AND+%22Methods%22%5BJournal%5D)">Barcoded cDNA library preparation for small RNA profiling by next-generation sequencing.</a> Methods. 58 (2), 164-170 (2012).</li>
<li>Loudig, O., Brandwein-Gensler, M., Kim, R. S., Lin, J., Isayeva, T., Liu, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Illumina+whole-genome+complementary+DNA-mediated+annealing%2C+selection%2C+extension+and+ligation+platform%3A+assessing+its+performance+in+formalin-fixed%2C+paraffin-embedded+samples+and+identifying+invasion+pattern-related+genes+in+oral+squamous+cell+carcinoma%5BTitle%5D)+AND+%22Hum.+Pathol%22%5BJournal%5D)">Illumina whole-genome complementary DNA-mediated annealing, selection, extension and ligation platform: assessing its performance in formalin-fixed, paraffin-embedded samples and identifying invasion pattern-related genes in oral squamous cell carcinoma.</a> Hum. Pathol. 42 (12), 1911-1922 (2011).</li>
</ol>

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 个单独的 100 ng FFPE RNA 样本) 的集合中。这一关键步骤允许 18 FFPE rna 样本一致接受所有后续的生物化学和酶步骤, 以准备小 RNA cDNA 文库。此外, 这一步通过提高小 RNA 分子的载体效应和促进颗粒观察和凝胶选择, 使降水和凝胶纯化的 RNA 数量最大化。该协议的另一个主要特点是, 在使用少量 FFPE RNA 时进一步强调了它的通用性, 即采用 PCR 反应来确定 cDNA 文库的最佳放大周期。这一步骤还提供了对图书馆扩增与底漆二聚体放大的动态的洞察, 这在制备大尺度 PCR 反应和纯化琼脂糖凝胶上的小 RNA 库方面是至关重要的。添加到本协议中的数据表明了这种方法在匹配的冷冻和 FFPE 标本之间的重现性, 同时也突出了它对较早的 FFPE RNA 样品的适用性, 这些标本存档了长达35年。
该协议是从其原始版本修改, 因此, 它是为制备小 RNA 库, 由低质量和数量的化学修饰和损害 FFPE rna。这一程序的一个方面被修改, 以成功地结扎和放大的小 rna 从 FFPE 标本是在 3 ' 条形码适配器结扎期间校准器鸡尾酒的数量, 其中输入减少到 0.026 nM, 以防止与低 abundancy 小 rna 结扎的竞争在 FFPE RNA 样品存在。对原程序的另一项重要修改是清除在页面凝胶上选择结扎的小 rna 时使用的所有放射性标记尺寸标记。这些核素大小标记的使用不仅限制了这种方法对使用无线电同位素认证的实验室的适用性, 而且也阻止了对凝胶上的 RNA 产品的观察。相反, 在这个优化的协议中, 大小标记在与库相邻的井中运行, 在页上凝胶上, 并直接与荧光染料可视化, 直接切除结扎的小 rna。重要的是, 荧光染料轻轻喷洒在凝胶上, 以防止 RNA 产品的扩散, 然后在一个蓝色的灯箱上形象化。随后, 作为预防措施和改善被结扎的小 rna 被包含在切除的页凝胶片断的扩散, 凝胶断路器管被用来均匀粉碎凝胶之前, 在一个 NaCl 溶液孵化过夜, 在4摄氏度下搅拌。所有这些步骤都经过仔细评估, 以使用少量的材料。
该协议适用于不同来源 (器官和机构) 的 FFPE RNA 样本, 这些样品已经储存了不同的时间。分析表明, 在使用这种优化过程中, 新鲜和冰冻标本对小 rna 的测序具有很高的重现性。使用保存长达35年的较旧的存档 FFPE 标本进行的额外分析进一步证明了该议定书对临床标本的广泛适用性。FFPE 标本的最小 FFPE RNA 输入要求被证明是 100 ng。这种低要求允许本协议适用于不同大小和可用性的各种病变, 但不允许对单细胞 FFPE RNA 进行分析。然而, 重要的是要注意的是, 这个优化的协议也被用于从循环外来体提取的总 RNA 与小于 1 ng 的输入, 并显示提供高度可重现的小 RNA 表达谱 (没有显示的数据)。这一低输入表明, FFPE 样品中的小 rna 虽然是原始新鲜组织的代表, 但比循环外来体中的总 RNA 存在的比例要低得多。
在最近的研究中, 将此优化的协议应用于临床分类 DCIS FFPE 标本, 发现通过定量 PCR 技术可以验证 cDNA 文库中的 miRNA 表达差异。这项工作表明, 使用 DCIS 病变的存档组织从不同的机构进行大规模的回顾性研究 [20]。这项研究还强调了这种 cDNA 文库在不同时间 (几周的间隔时间) 进行的健壮性, 而不影响化验的重现性和敏感性。
考虑到大量临床分类存档的 FFPE 标本, 该优化协议为在大规模回顾性分析中制备 cDNA 文库和 miRNA 生物标志物的潜在鉴定提供了有力的工具。与癌症发展相关的21。

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)。切除后的凝胶片在 NaCl 溶液中孵化, 洗脱结扎的 RNA 分子。第二天, 洗脱 RNA 沉淀, 并执行 5 ' 适配器结扎。然后, 5 ' 适配器结扎小 RNA 分子迁移和分离在12% 丙烯酰胺凝胶, 再次迁移 RNA 大小标记寡核苷酸允许大小切除的小 rna 包含 3 ' 条形码寡核苷酸和 5 ' 适配器 (图 3)。该切除凝胶是在一夜之间的 NaCl 溶液孵化, 以允许洗脱的 RNA。第二天, 结扎的小 RNA 分子沉淀, 颗粒在无 RNase 水中悬浮, 其次为反转转录;整除的 cDNA 分子经历了一个先导性 PCR 反应 (图 4A)。使用同一输入的 cDNA 文库的大型 pcr 反应建立和评估2.5% 琼脂糖凝胶, 以验证所有反应是充分 PCR 扩增 (图 4B), 在池和隔夜乙醇沉淀之前。第二天, 放大 cDNA 库包含所有18个单独的图书馆为18个独特的 FFPE RNA 标本, 迁移到2.5% 琼脂糖凝胶, 和最高 PCR 波段, 运行在 100 nt 是切除和纯化 (图 4C)。然后在高灵敏度的 DNA 芯片 (图 5) 上对 cDNA 文库进行纯化, 以确定纯化的 pcr 产物不含过量的底漆脂肪酸或 pcr 反应的其他副产品。然后在高通量测序系统上对 PCR 产物进行分析。适配器修剪和生成18个单独的文件为每一个18标本使用 RNAworld 管道 (访问是提供给我们的托马斯博士 Tuschl)。然后进行 Biostatistical 分析, 评价 FFPE RNA 标本的 miRNA 含量。
为了验证这个优化的过程, 匹配的新鲜冰冻和 FFPE 乳腺肿瘤标本用于分析 (图 6)。选择了两种类似的侵袭性导管乳腺癌 (IDC) 肿瘤, 以评估该程序的敏感性, 并确定两个新鲜冰冻组织之间发现的 miRNA 表达差异是否也可以在匹配的存档 FFPE 中检测到。RNA 标本。在本实验中, 对2新鲜冷冻的总 RNA 的质量和匹配的两个 FFPE rna 样本进行了评估 (图 6A)。正如预期的, 与匹配的新鲜冷冻 rna 相比, FFPE 标本的 RNA 大小和质量严重下降 (比较车道1和 3, 车道2和 4)。其中一个 FFPE RNA 已存档在 rt 4 年 (侵袭性乳腺癌 1, (IBC1)), 另一人已存档在 rt 8 年 (IBC2), 而新鲜冰冻对应储存在-80 摄氏度。在单个库中分析了四个单独的 RNA 样本, 使用了4个单独的条形码, 并且 miRNA 读取分布图显示在图 6B中。两个顶部板显示两个新鲜的冰冻肿瘤 rna 和它们的特异 FFPE RNA 标本 miRNA 表达的相关性。配对新鲜冷冻和 FFPE miRNAs 之间的地块表明, cDNA 文库的制备提供了良好的重现性, 因为在不同的标本中检测到的 miRNAs 之间可以观察到较高的相关性 (冷冻 vs FFPE)。两个较低的面板显示 miRNA 表达数据之间的关系, 两个不同的冰冻肿瘤和两个不同的 FFPE 肿瘤。如图 6C所示, 两个新鲜冰冻肿瘤之间发现的 miRNA 表达差异与两个匹配的 FFPE 肿瘤标本之间检测到的差异相关。新鲜冰冻和 FFPE 肿瘤均可检测 miRNA 表达差异的意义。
为了进一步评估这种方法的敏感性, 使用了来自12个存档 FFPE 标本和4个新鲜冰冻 RNA 标本的 miRNA 表达数据 (图 6D)。16种不同的 RNA 标本分别为 3 ' 条形码, 全部用于制备单个 cDNA 文库。该库中使用的 rna 样本包括两个乳腺细胞系, MCF10A (正常样细胞线) 和 MCF7 (乳腺癌细胞系) 与 RNA 从新鲜细胞和他们的存档 FFPE 对应23, 匹配新鲜冷冻和 FFPE RNA 样品从两个乳腺癌样本独立分析 (IBC1 和 IBC2 在图 6A和6B), 并匹配新鲜冰冻和 FFPE RNA 样本从正常宫颈标本 (Cx)。此外, 存档的 FFPE 标本从正常的 (正常 Br1, 正常 Br2, 正常 Br3) 和癌症乳房组织 (ibc 5, ibc 6, 和 ibc 7), 没有他们新鲜冰冻对应的分析。正如观察到的热图, 无论新鲜冰冻或 FFPE RNA 起源, miRNA 表达谱的同一细胞 (MCF10 或 MCF7), 或相同的组织 (IBC1, IBC2, 或 Cx) 聚集在一起。此外, 正如在无监督的群集上指出的, 从左到右, 正常的乳房细胞和组织聚集在一起, 而乳腺肿瘤和肿瘤细胞聚集在右侧。宫颈组织显示不同的 miRNA 表达谱, 聚集在热图的右侧。
考虑到大多数临床存档的 FFPE 标本没有新鲜冰冻的对应, 但它们可以在不同贮存期后检索, 因此寻求确定最佳 cDNA 文库准备协议是否适用, 并重现与越来越老的 FFPE 标本。如图 7所示, 获得了存档了18、20、22、27、30和35年的 FFPE 组织中的 miRNA 表达式配置文件。rna 被提取使用优选的同时 rna 或脱氧核糖核酸做法21和皮带 RNA 整除数从每个单独 FFPE 标本在同一天被准备了并且存放了在-80 °c 在图书馆准备之前。共对9种不同的 FFPE 标本进行了重复分析, 其中每个 RNA 整除在同一图书馆内以不同的条形码寡核苷酸 (18 码总) 结扎。这项实验在连续两周 (1 周和2周) 重复进行。这允许在单个库中使用两个不同的条形码, 并在两个不同的库之间用一周时间间隔来评估 cDNA 文库的制备重现性。正如在图 7中所观察到的, 无论标本的年龄或库准备周如何, 相关系数仍保持在0.96 以上;因此, 优化的 cDNA 文库准备协议为 FFPE 标本的重现性分析提供了强有力的工具, 无论其存档时间如何, 例如, 35 岁的存档 FFPE RNA (见乳房 #9) 显示高重现性措施相当于20岁的 FFPE RNA 样本 (见乳房 #3)。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57471/57471fig1.jpg"> 图 1: 寡核苷酸.本文介绍了所有的寡核苷酸序列、相应的化学修饰和在本协议中使用的浓度。化学修饰的类型在修改框和寡核苷酸序列中显示的缩写修改中描述。校准剂鸡尾酒显示10个单独 RNA 寡核苷酸校准器名单, 在包含载体寡核苷酸 (0.5 µM) 的解决方案中悬浮。十八 3 ' 条形码寡核苷酸适配器是详细的灰色阴影在条形码的序列。详细介绍了 5 ' 适配器的 RNA 序列, 以及 3 ' pcr 和 5 ' pcr 引物的 DNA 序列。提供了两个大小标记寡核苷酸的 RNA 序列, 即协议中引用的 19 nt-3 适配器和 24 nt-3 适配器大小标记。所有的 DNA 和 RNA 寡核苷酸都是商业购买。<a href="/files/ftp_upload/57471/57471fig1large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/57471/57471fig2.jpg"> 图 2: 页面纯化 3 ' 条形码 RNA 样品.在共用和沉淀18条形码 rna 样品后, 悬浮 rna 颗粒通过电泳在15% 丙烯酰胺凝胶上迁移和分离 (见 8)。红色正方形突出了包含 3 ' 条形码 microRNAs 的区域, 它被切除与手术刀刀片并且转移入一个核酸酶无渗离心管。共有四口井, 其中两个包含 19 nt-3 适配器和两个包含 24 nt-3 ' 适配器是设置了一个很好的, 在图书馆的每一侧 (见水井 5, 6, 10, 11, 分别)。作为测试 T4 RNA 连接酶1和为净化的结扎尺寸标记将完成第二天, 结扎反应包含 19 nt-3 ' 适配器大小标记与 5 ' 适配器和 24 nt-3 ' 适配器大小标记与 5 ' 适配器运行在井2d 3, 分别。黄色方块显示的切除带代表结扎大小标记 RNA 寡核苷酸与 5 ' 适配器。这些带被纯化, 沉淀, 并运行在12% 页净化 (请参见下面的图 3 )。井1和13包含 20 nt 大小梯子, 帮助确定被结扎的产品的预期的大小。<a href="/files/ftp_upload/57471/57471fig2large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/57471/57471fig3.jpg"> 图 3: 在 5 ' 适配器结扎后净化库的页面纯化.纯化的 RNA 库被结扎与 5 ' 适配器和预期的产品大小被切除从12% 页使用结扎大小标记 (参见红色正方形)。T4 rna 结扎的产品在 19 nt-3 ' 适配器和 5 ' 适配器 (井4和 9) 之间, 并且24个 nt-3 ' 适配器和 5 ' 适配器 (井5和 10) 之间平行地运行, 在包含 RNA 图书馆的井的每边。最高波段 (见白星号) 是 5 ' 适配器结扎的产品, 并用作用于包含 5 ' 适配器结扎 RNA 库的凝胶带切除的指南。在上一页上提纯的结扎尺寸标记结扎产品, 运行在3井。如在水井1和12中所观察到的, 20 nt 大小的梯子也被运行以验证 RNA 库的预期大小。<a href="/files/ftp_upload/57471/57471fig3large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/57471/57471fig4.jpg"> 图 4: 初步 PCR 和大规模放大 cDNA 文库.用2.5% 琼脂糖凝胶在溴溴化物的存在下, 对 PCR 产物的大小和配比进行了评价。(A) 在条形码 RNA 库的反向转录后, 使用 5 ' 和 3 ' pcr 引物在单一的 pcr 反应中放大了 cDNA 文库的整除。整除数的 PCR 反应得到了 10, 12, 14, 16, 18 和20循环和迁移在一个2.5% 琼脂糖凝胶。在井1和7之间观察, cDNA 库和适配器脂肪酸的存在是指数可见的 (参见绿色矩形)。对于该文库, 选择用于扩增 cDNA 文库的 PCR 循环为15。(B) 此示意图显示了不同的寡核苷酸的位置和长度, 以及由此产生的 RNA 和 DNA 产品, 它们可在页面上和琼脂糖凝胶上识别。(C) 对6种大型 PCR 反应 (在A中确定) 的整除数分别在2.5% 琼脂糖凝胶上进行了分析 (见水井3至 8)。这两种类型的 PCR 产品, 即图书馆 (上带) 和引物脂肪酸 (下带) 是可见的在这个凝胶 (见绿色矩形)。无 cDNA 的空白 pcr 反应不显示 pcr 放大 (参见井 2)。20 nt 大小的梯子允许验证产品尺寸 (见 1)。(D) 在蓝色光 transilluminator 上的凝胶图像, 包含聚合 PCR 反应的2.5% 琼脂糖凝胶在两个相邻的井中运行。观察到, 两个井中最高的波段是在预期的库大小内, 适配器二聚体带位于下面。用凝胶萃取试剂盒对上 PCR 带进行切除纯化。<a href="/files/ftp_upload/57471/57471fig4large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/57471/57471fig5.jpg"> 图 5: 对纯化 PCR 扩增 DNA 库的评估.采用高灵敏度的 DNA 芯片对微流体平台进行 PCR 扩增 cDNA 文库的小整除 (1 µL) 分析。(A) 此面板显示用于校准仪器的尺寸标记的迁移。(B) 此面板显示纯化 PCR 扩增 cDNA 文库的迁移。最高峰值以100的 bp (见星号) 的大小来衡量, 并代表 cDNA 文库。该仪器对 72 bp 的小峰进行了评估, 表示底漆适配器脂肪酸。基于微流体平台检测到的 100 bp 峰值显示了扩增 cDNA 文库的估计大小, 其中包含18个单独的条形码 RNA 样本, 用于随后的高通量测序系统。<a href="/files/ftp_upload/57471/57471fig5large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/57471/57471fig6.jpg"> 图 6: 使用匹配的新鲜冷冻和福尔马林固定石蜡嵌入标本的 cDNA 文库制备和下一代测序.(A) 从匹配的新鲜冰冻和 FFPE 侵袭性导管癌 (IDC) 肿瘤中提取的总 rna, 在一个基于微流体的平台上对总 rna 芯片进行了分析。(B) 匹配的新鲜冷冻和 FFPE 标本 (IBC 1 和 IBC2) 的总 RNA 接受了 cDNA 文库准备协议, 绘制了 miRNA 测序数据。(C) DifferentialmiRNA IBC1/IBC2 标本之间的表达, 以及冷冻和 FFPE 配对样品之间的相关性。miRNA 表达式以每百万 (CPM) 的对数计数显示, 从高到低读取 (红色到蓝色)。两个组织对的表达差异的意义, 每 miRNA, 是显示的灰色圆圈, 高和低表达 miRNAs 确定在新鲜和 FFPE 样品。(D) 从乳腺细胞系的新鲜和 FFPE rna 标本 (MCF10A 和 MCF7)、人侵袭性乳腺癌 (IBC 1 和 IBC2)、宫颈组织 (Cx)、存档正常乳腺组织 (正常 Br1、正常 Br2 和正常 Br 3) 中提取的总 rna, 以及归档侵袭性乳腺癌 (ibc 5, ibc 6 和 IBC7) 在同一运行中接受了 cDNA 文库的准备。在库中检测到的 miRNAs 数据将显示在热映射配置中。此数字已从 Loudig et . 中进行了修改。20<a href="/files/ftp_upload/57471/57471fig6large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 7" class="xfigimg" src="/files/ftp_upload/57471/57471fig7.jpg"> 图 7: 使用较旧的存档 FFPE 标本进行复制的 cDNA 库准备和 miRNA 表达式相关性.总 RNA 从 18, 20, 22, 27, 30 和35年老归档的 FFPE 乳房组织标本接受了我们优选的 cDNA 文库准备协议。从9种不同的 FFPE 标本中复制 RNA 样本, 用不同的 3 ' 条形码寡核苷酸结扎, 并在1周 (w1) 的同一图书馆内进行分析。同样的实验在一周的时间间隔内重复 (2 周或 w2)。重复 miRNA 表达数据的重现性措施在同一存档的 RNA 标本之间显示在 heatmaps 和相关系数评估在0.93 和0.99 之间。此数字已从 Loudig et . 中进行了修改。20<a href="/files/ftp_upload/57471/57471fig7large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>

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

Nanyang Technological University

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

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 ...

科研

遗传学

Cerebrospinal Fluid MicroRNA Profiling Using Quantitative Real Time PCR

MicroRNAs (miRNAs) constitute a potent layer of gene regulation by guiding RISC to target sites located on mRNAs and, consequently, by modulating their translational repression. Changes in miRNA expression have been shown to be involved in the development of all major complex diseases. Furthermore, recent findings showed that miRNAs can be secreted to the extracellular environment and enter the bloodstream and other body fluids where they can circulate with high stability. The function of such circulating miRNAs remains largely elusive, but systematic high throughput approaches, such as miRNA profiling arrays, have lead to the identification of miRNA signatures in several pathological conditions, including neurodegenerative disorders and several types of cancers. In this context, the identification of miRNA expression profile in the cerebrospinal fluid, as reported in our recent study, makes miRNAs attractive candidates for biomarker analysis.
There are several tools available for profiling microRNAs, such as microarrays, quantitative real-time PCR (qPCR), and deep sequencing. Here, we describe a sensitive method to profile microRNAs in cerebrospinal fluids by quantitative real-time PCR. We used the Exiqon microRNA ready-to-use PCR human panels I and II V2.R, which allows detection of 742 unique human microRNAs. We performed the arrays in triplicate runs and we processed and analyzed data using the GenEx Professional 5 software.
Using this protocol, we have successfully profiled microRNAs in various types of cell lines and primary cells, CSF, plasma, and formalin-fixed paraffin-embedded tissues.

We describe a protocol of real time PCR to profile microRNAs in the cerebrospinal fluid (CSF). With the exception of RNA extraction protocols, the procedure can be extended to RNA extracted from other body fluids, cultured cells, or tissue specimens.

脑脊液的microRNA分析采用实时定量PCR技术

MicroRNAs (miRNAs) constitute a potent layer of gene regulation by guiding RISC to target sites located on mRNAs and, consequently, by modulating their ...

医学

A Rapid High-throughput Method for Mapping Ribonucleoproteins (RNPs) on Human pre-mRNA

Sequencing RNAs that co-immunoprecipitate (co-IP) with RNA binding proteins has increased our understanding of splicing by demonstrating that binding location often influences function of a splicing factor.  However, as with any sampling strategy the chance of identifying an RNA bound to a splicing factor is proportional to its cellular abundance.  We have developed a novel in vitro approach for surveying binding specificity on otherwise transient pre-mRNA. This approach utilizes a specifically designed oligonucleotide pool that tiles across introns, exons, splice junctions, or other pre-mRNA.  The pool is subjected to some kind of molecular selection. Here, we demonstrate the method by separating the oligonucleotide into a bound and unbound fraction and utilize a two color array strategy to record the enrichment of each oligonucleotide in the bound fraction. The array data generates high-resolution maps with the ability to identify sequence-specific and structural determinates of ribonucleoprotein (RNP) binding on pre-mRNA.  A unique advantage to this method is its ability to avoid the sampling bias towards mRNA associated with current IP and SELEX techniques, as the pool is specifically designed and synthesized from pre-mRNA sequence. The flexibility of the oligonucleotide pool is another advantage since the experimenter chooses which regions to study and tile across, tailoring the pool to their individual needs.  Using this technique, one can assay the effects of polymorphisms or mutations on binding on a large scale or clone the library into a functional splicing reporter and identify oligonucleotides that are enriched in the included fraction.  This novel in vitro high-resolution mapping scheme provides a unique way to study RNP interactions with transient pre-mRNA species, whose low abundance makes them difficult to study with current in vivo techniques.  



A Rapid High-throughput Method for Mapping Ribonucleoproteins RNPs on Human pre-mRNA

Due to the transient nature of pre-mRNA, it can be difficult to isolate and study in vivo. Here, we present a novel in vitro approach to investigate RNA-protein interactions using a synthetic oligo pool that tiles across selected regions of pre-mRNA.

一种快速高通量的方法映射核糖核蛋白（RNPs）人类的前体mRNA

Sequencing RNAs that co-immunoprecipitate (co-IP) with RNA binding proteins has increased our understanding of splicing by demonstrating that binding ...

生物学

miRNA Expression Analyses in Prostate Cancer Clinical Tissues

A critical challenge in prostate cancer (PCa) clinical management is posed by the inadequacy of currently used biomarkers for disease screening, diagnosis, prognosis and treatment. In recent years, microRNAs (miRNAs) have emerged as promising alternate biomarkers for prostate cancer diagnosis and prognosis. However, the development of miRNAs as effective biomarkers for prostate cancer heavily relies on their accurate detection in clinical tissues. miRNA analyses in prostate cancer clinical specimens is often challenging owing to tumor heterogeneity, sampling errors, stromal contamination etc. The goal of this article is to describe a simplified workflow for miRNA analyses in archived FFPE or fresh frozen prostate cancer clinical specimens using a combination of quantitative real-time PCR (RT-PCR) and in situ hybridization (ISH). Within this workflow, we optimize the existing methodologies for miRNA extraction from FFPE and frozen prostate tissues and expression analyses by Taqman-probe based miRNA RT-PCR. In addition, we describe an optimized method for ISH analyses formiRNA detection in prostate tissues using locked nucleic acid (LNA)- based probes. Our optimized miRNA ISH protocol can be applied to prostate cancer tissue slides or prostate cancer tissue microarrays (TMA).

Here we describe a simplified protocol for microRNA (miRNA) expression analyses in archived Formalin-Fixed, Paraffin-Embedded (FFPE) or fresh frozen prostate cancer (PCa) clinical tissues employing quantitative real-time PCR (RT-PCR) and in situ hybridization (ISH).

在前列腺癌的临床组织中miRNA的表达分析

A critical challenge in prostate cancer (PCa) clinical management is posed by the inadequacy of currently used biomarkers for disease screening, ...

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

MicroRNA In situ Hybridization for Formalin Fixed Kidney Tissues

In this article we describe a method for colorimetric detection of miRNA in the kidney through in situ hybridization with digoxigenin tagged microRNA probes. This protocol, originally developed by Kloosterman and colleagues for broad use with Exiqon miRNA probes1, has been modified to overcome challenges inherent in miRNA analysis in kidney tissues. These include issues such as structure identification and hard to remove residual probe and antibody. Use of relatively thin, 5 mm thick, tissue sections allowed for clear visualization of kidney structures, while a strong probe signal was retained in cells. Additionally, probe concentration and incubation conditions were optimized to facilitate visualization of microRNA expression with low background and nonspecific signal. Here, the optimized protocol is described, covering the initial tissue collection and preparation through the mounting of slides at the end of the procedure. The basic components of this protocol can be altered for application to other tissues and cell culture models.

MicroRNA In situ Hybridization for Formalin Fixed Kidney Tissues

This article describes an in situ hybridization protocol optimized for colormetric detection of microRNA expression in formalin fixed kidney sections.

微RNA<em&gt;原位</em&gt;杂交的福尔马林固定的肾组织

In this article we describe a method for colorimetric detection of miRNA in the kidney through in situ hybridization with digoxigenin tagged microRNA ...

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 ...

FFPE Tissue Pretreatment for RNA CISH: A Procedure to Process Formalin-Fixed Paraffin-Embedded Tissue Sections for RNA Chromogenic In Situ Hybridization

Source: Outh-Gauer, S. et al. <a href="https://www.jove.com/video/59422" target="_blank">Chromogenic In Situ Hybridization as a Tool for HPV-Related Head and Neck Cancer Diagnosis.</a> J. Vis. Exp. (2019)
This video demonstrates the steps involved in the pretreatment of FFPE tissue specimens for RNA chromogenic in situ hybridization. The pretreatment process helps maintain nucleic acid integrity within the cells and overall tissue morphology.

RNA CISH 的 FFPE 组织预处理:处理福尔马林固定石蜡包埋的组织切片以进行 RNA 显色原位杂交的程序

Source: Outh-Gauer, S. et al. Chromogenic In Situ Hybridization as a Tool for HPV-Related Head and Neck Cancer Diagnosis. J. Vis. Exp. (2019)
This video ...