Name: retrospective microrna sequencing: complementary dna library preparation protocol using formalin-fixed paraffin-embedded rna specimens
Uploaded: 2018-05-05T13:00:00
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。

1. 所有试剂和底漆的制备
<ol><li>按照图 1中的说明, 订购所有底漆和适配器。</li>
	<li>
准备校准鸡尾酒的股票, 这将是在每个单独结扎的尖刺。
	<ol>
<li>将载波寡核苷酸 (图 1) 并用重悬为0.5 µM, RNase 无水。使用0.5 µM 载波寡核苷酸解决方案, 将10校验和寡核苷酸 (图 1) 并用重悬为100µM。</li>
		<li>将渗离心中的每个校准器?...

<ol>
	<li>Peir&#243;-Chova, L., Pe&#241;a-Chilet, M., L&#243;pez-, J. A., Garc&#237;a-Gimenez, J. L., Alonso-Yuste, E., Burgues, O., 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=High+stability+of+microRNAs+in+tissue+samples+of+compromised+quality.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Excavation+of+a+buried+treasure--DNA,+mRNA,+miRNA+and+protein+analysis+in+formalin+fixed,+paraffin+embedded+tissues.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MicroRNA+Stability+in+Postmortem+FFPE+Tissues:+Quantitative+Analysis+Using+Autoptic+Samples+from+Acute+Myocardial+Infarction+Patients.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hsa-miR-375+is+differentially+expressed+during+breast+lobular+neoplasia+and+promotes+loss+of+mammary+acinar+polarity.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Robust+global+micro-RNA+profiling+with+formalin-fixed+paraffin-embedded+breast+cancer+tissues.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+array-based+analysis+of+microRNA+expression+comparing+matched+frozen+and+formalin-fixed+paraffin-embedded+human+tissue+samples.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multi-platform+analysis+of+microRNA+expression+measurements+in+RNA+from+fresh+frozen+and+FFPE+tissues.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+microRNA+deep+sequencing+of+matched+formalin-fixed+paraffin-embedded+and+fresh+frozen+cancer+tissues.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MicroRNA+expression+profiling+outperforms+mRNA+expression+profiling+in+formalin-fixed+paraffin-embedded+tissues.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MicroRNA+expression+in+formalin-fixed+paraffin+embedded+tissue+using+real+time+quantitative+PCR:+the+strengths+and+pitfalls.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tissues+from+routine+pathology+archives+are+suitable+for+microRNA+analyses+by+quantitative+PCR.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+miRNA+expression+analysis+using+fluidigm+microfluidics+dynamic+arrays.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improved+microRNA+quantification+in+total+RNA+from+clinical+samples.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Next-generation+sequencing+and+microarray-based+interrogation+of+microRNAs+from+formalin-fixed,+paraffin-embedded+tissue:+preliminary+assessment+of+cross-platform+concordance.">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="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MiRNAs+and+cancer.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MicroRNA+expression+profiles+classify+human+cancers.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MicroRNAs:+potential+biomarkers+for+cancer+diagnosis,+prognosis+and+targets+for+therapy.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Micro-RNAs+as+diagnostic+or+prognostic+markers+in+human+epithelial+malignancies.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Robust+global+microRNA+expression+profiling+using+next-generation+sequencing+technologies.">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/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. A., Mihailovi&#263;, A., Pena, J. T., Tuschl, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Barcoded+cDNA+library+preparation+for+small+RNA+profiling+by+next-generation+sequencing.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=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.">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>

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&#8482; 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 ...

Research

JoVE Journal

Genetics

Methyl-binding DNA capture Sequencing for Patient Tissues

为患者组织甲基结合DNA序列捕获

Methylation is one of the essential epigenetic modifications to the DNA, which is responsible for the precise regulation of genes required for stable ...

科研

遗传学

Mapping RNA-RNA Interactions Globally Using Biotinylated Psoralen

使用生物素化补骨脂素全球定位RNA-RNA相互作用

Knowing how RNAs interact with themselves and with others is key to understanding RNA based gene regulation in the cell. While examples of RNA-RNA ...

Assessment of DNA Contamination in RNA Samples Based on Ribosomal DNA

基于核糖体 dna 的 RNA 样本 dna 污染评价

One method extensively used for the quantification of gene expression changes and transcript abundances is reverse-transcription quantitative real-time ...

A Universal Protocol for Large-scale gRNA Library Production from any DNA Source

一种从任意 DNA 源进行大规模 gRNA 库生产的通用协议

The popularity of the CRISPR/Cas9 system for both genome and epigenome engineering stems from its simplicity and adaptability. An effector (the Cas9 ...

Robust DNA Isolation and High-throughput Sequencing Library Construction for Herbarium Specimens

植物标本标本的鲁棒 DNA 分离与高通量测序库建设

Herbaria are an invaluable source of plant material that can be used in a variety of biological studies. The use of herbarium specimens is associated with ...

Rare Event Detection Using Error-corrected DNA and RNA Sequencing

基于纠错 DNA 和 RNA 测序的稀有事件检测方法

Conventional next-generation sequencing techniques (NGS) have allowed for immense genomic characterization for over a decade. Specifically, NGS has been ...

Ultralow Input Genome Sequencing Library Preparation from a Single Tardigrade Specimen

单缓步动物标本的超低输入基因组测序库制备

Tardigrades are microscopic animals that enter an ametabolic state called anhydrobiosis when facing desiccation and can return to their original state ...

Sequencing of mRNA from Whole Blood using Nanopore Sequencing

纳米测序全血 mRNA 测序

Sequencing in remote locations and resource-poor settings presents unique challenges. Nanopore sequencing can be successfully used under such conditions, ...

Single-cell RNA Sequencing and Analysis of Human Pancreatic Islets

人类胰腺胰岛的单细胞RNA测序与分析

Pancreatic islets comprise of endocrine cells with distinctive hormone expression patterns. The endocrine cells show functional differences in response to ...

Identification of Footprints of RNA:Protein Complexes via RNA Immunoprecipitation in Tandem Followed by Sequencing (RIPiT-Seq)

Identification of Footprints of RNA:Protein Complexes via RNA Immunoprecipitation in Tandem Followed by Sequencing RIPiT-Seq

RNA足迹的识别:通过RNA免疫沉淀在串联中的蛋白质复合物,然后测序(RIPiT-Seq)

RNA immunoprecipitation in tandem (RIPiT) is a method for enriching RNA footprints of a pair of proteins within an RNA:protein (RNP) complex. RIPiT ...