我们感谢罗杰·埃里克森博士对他的支持和帮助准备手稿。 这项工作是由美国国家癌症研究所美国国立卫生研究院的支持 （批准号RO1CA177984; RO1CA138642），对前列腺癌（BX001604）VA计划项目。

福尔马林固定，石蜡包埋（FFPE）的或新鲜冷冻的前列腺癌样品从SFVAMC获得。样品从前列腺癌患者在谁SFVAMC接受前列腺癌根治术。书面知情同意，所有患者和研究经加州大学旧金山分校人类研究委员会。可替代地，前列腺癌组织的微阵列从商业来源采购并用于miRNA的分析由ISH。收集临床病理及随访资料分析，前列腺癌患者。 1.组织样本<ol><li>切的前列腺癌组织样品成使用切片机和染色用H＆E按照制造商的协议10微米的部分。 </li><li>检查染色切片用于前列腺癌病灶的标识以及邻近的正常腺上皮。 注:委员会认证的病理学家应检讨H＆E染色切片和马的rk为肿瘤和正常区域。使用标记幻灯片中的miRNA的后续部分导游分析肿瘤与正常区域。 </li></ol> 2. miRNA的表达分析定量实时荧光定量PCR 注意:此流程包括以下步骤:激光捕获显微切割，总RNA（包括miRNA与mRNA）的分离，使用如在以下部分中详细描述的的TaqMan微小RNA表达测定成熟miRNA的含量测定。 <ol><li> RNA提取<ol><li>前列腺癌FFPE组织提取的miRNA <ol><li>激光捕获显微切割（LCM） 注:执行步骤2.1.1.1.1- 2.1.1.1.4在通风橱。 <ol><li>对于LCM准备组织切片（10微米的部分）。 Deparrafinize组织切片在二甲苯浸泡（2次），每10分钟，然后通过将幻灯片的每个在梯度乙醇（100％，95％，90％，80％，70％）5分钟，然后用蒸馏水补湿组织水（5分钟）。 </li><li>以下补液，染色切片用苏木精30秒，然后用水。 </li><li>地方的组织在梯度乙醇（70％，95％90％，80％，70％）（每次5分钟）和二甲苯（5分钟）。 </li><li>在通风橱中干燥载玻片，然后将在LCM的仪器为显微切割。 </li><li>按照制造商的说明24执行microdissections与LCM系统。使用病理学家标记的幻灯片作为指南，前列腺癌病灶的标识以及邻近的正常组织。使用激光束在10-20微米的直径脉冲70-90毫瓦的功率。 </li><li>用红外激光脉冲的兴趣到LCM上限捕捉区域。从2-5个盖帽为每个样品的miRNA萃取结合细胞。为确保提取的RNA完整性，立即处理捕获的细胞miRNA的提取。 </li><li>备选地，裂解细胞中miRNA的提取缓冲液（根据制造商的协议）和STO再裂解液在-80ºC。 </li></ol></li><li> miRNA的提取和分析，从LCM显微组织<ol><li>从使用商业试剂盒按照制造商的说明，显微切割的FFPE组织中提取总RNA。 注:RNA的激光捕获miscrodissection的收益率通常较低。因此，RNA被洗脱到小体积（20-30微升）和用于使用以下制造商的说明的Taqman微小RNA表达分析表达谱（2.2节中也有描述）。优化的输入核糖核酸为实时PCR检测成熟miRNA的表明5微升洗脱的RNA是最佳的每个miRNA特异性逆转录反应。作为内源对照，RNU48 / RNU24被使用。对于对照反应，2微升洗脱的RNA用于逆转录反应。 </li></ol></li></ol></li><li>前列腺癌冷冻组织提取的miRNA <ol><li> Homoge通过使用研钵和研杵研磨该组织在液氮中冷冻nize切除前列腺组织。使用冷却的刮刀转移匀浆组织到一个离心管中。通过在液氮中，以便匀浆组​​织浸渍可以容易地转移保持砂浆/杵和抹刀在相同的温度。 </li><li>添加商业的异硫氰酸胍 - 苯酚试剂（1毫升/ 0.1克​​组织）的均质化的组织和在室温下孵育5分钟。 注意:在商业异硫氰酸胍 - 酚试剂均化组织可以储存在-80ºC数月。 </li><li>加三氯甲烷匀浆（0.3毫升氯仿/毫升），并剧烈摇晃30秒。孵育的样品在RT 3-5分钟，随后离心以10,000×g离心20分钟，在4ºC。 </li><li>上层水相转移到一个新的无菌无RNase 1.5毫升试管中。 </li><li>添加异丙醇等体积。混合和孵育10英里n将在室温然后离心在12,000×g离心20分钟，在4℃到沉淀的RNA。 </li><li>吸出上清液，并用1mL 70％乙醇洗涤RNA沉淀。离心机在12,000rpm离心10分钟，在4ºC。 </li><li>小心吸出上清液。干燥核糖核酸粒料在RT 5-10分钟。在50-100微升无核酸酶水溶解RNA。 </li><li>使用NanoDrop分光光度计进行的RNA定量，并确定与一个生物分析RNA的完整性。调整RNA浓度为10毫微克/微升。用10-50毫微克的RNA的miRNA-特异基因反应，接着通过实时PCR分析（2.2节）。 </li></ol></li></ol></li><li>定量实时PCR检测miRNA表达分析 注:测定成熟miRNA使用两步骤RT-PCR方案如下所述。 <ol><li>反转录（RT） <ol><li>反向使用的miRNA逆转录试剂盒按照制造商的说明由RNA转录的cDNA。使用10-50纳克的总RNA与miRNA的从微RNA测定法和RT试剂盒特异性引物。使用RNU48 / RNU24 / RNU6B作为对照。稀释的cDNA 1:5至1:10（取决于所分析的miRNA的丰度）并如以下步骤描述在实时PCR检测使用。 </li></ol></li><li>实时PCR检测成熟miRNA的<ol><li>扩增PCR产物利用微RNA检测具有快速通用预混按照制造商的说明cDNA样品。规范化样品RNU48 / RNU24 / RNU6B控制。使用比较Ct（阈值循环）方法来计算上快速实时PCR系统中的基因表达的相对变化。一式三份每个样品分析。 </li></ol></li></ol></li></ol> 3. miRNA的表达分析通过原位杂交（ISH） <ol><li>组织的前处理<ol><li>切从使用切片机FFPE前列腺癌组织块5微米的部分。 </li><lI&gt;孵育载玻片在56℃下1小时固定的组织切片。 注意:幻灯片可以存储在RT几个星期的miRNA分析。 </li><li>浸泡载玻片用二甲苯（2×），每次15分钟，Deparrafinize组织。 </li><li>通过将载玻片5分钟，在每一个分级的乙醇（100％（2次），95％，90％，80％，70％），然后加入蒸馏水（5分钟）再水合组织。 </li><li>固定用溶于PBS的4％多聚甲醛的幻灯片在室温下20分钟。 </li><li>在室温下洗涤​​用PBS（2×）的幻灯片每次5分钟。 </li><li>治疗的幻灯片用10微克/毫升蛋白酶K在37ºC为在预热的蛋白酶K缓冲液10分钟（5毫摩尔Tris-HCL pH7.4的1mM EDTA中，1毫摩尔NaCl）。 </li><li>下列蛋白酶K处理，冲洗用0.2％的甘氨酸的幻灯片在PBS中持续30秒。 </li><li>在PBS（1×），在室温下5分钟洗幻灯片。 </li><li>固定用溶于PBS的4％多聚甲醛的幻灯片15分钟。 </li><li>冲洗幻灯片PBS 5分钟。 </li></ol></li><li>杂交<ol><li>预杂交用3-4小时，在湿盒预杂交溶液在55ºC幻灯片。使用浸泡在50％甲酰胺/ 50％5×SSC组织实验室湿巾以保持腔室加湿。 </li><li>使用5&#39;洋地黄毒苷标记的探针，在20-50纳米杂交缓冲液中的浓度。稀的miRNA特异性探针（20-50纳米）和小核RNA U6对照探针（20纳米），热在90ºC4分钟，将其放置在冰上，并加入到冰冷的杂交缓冲液（2-4毫微克/微升）。 </li><li>除去预杂交液，加入杂交液（探针+杂交缓冲液，每幻灯片100微升），孵育12-16小时，在探针特异性杂交​​的温度（杂交温度= Tm为探针 - 21℃）。在湿润的腔室（50％甲酰胺/ 50％5×SSC）的执行杂交。 </li></ol></li><li>洗涤步骤<ol><li> 10分钟，45ºC洗涤用2×SSC的幻灯片。 </li><li>洗吨他幻灯片1.5倍SSC在45ºC10分钟。 </li><li>在37ºC每次20分钟，洗涤0.2×SSC（2×）的幻灯片。 </li><li>孵育载玻片用1×阻断溶液1-2小时，在室温下</li></ol></li><li>发现<ol><li>孵育载玻片以1:100 PBS稀释的AP缀合的抗洋地黄毒苷抗体为1-4小时或过夜，在4ºC。 </li><li>在室温下洗涤​​用PBS（3×）的幻灯片，每次10分钟。 </li><li>洗载玻片（2次）与碱性磷酸酶缓冲液（100mM的Tris pH为9.5，50mM的MgCl 2的，100 mM氯化钠，0.1％吐温-20）在室温下，每次5分钟。 </li><li>孵育在BM紫AP底载玻片在黑暗中在RT 1-20小时。 </li><li>冲洗载玻片用PBS含有0.1％Tween-20和洗涤（2×）水。 </li><li>安装使用含水封装介质的幻灯片，并在显微镜下检查。 </li></ol></li></ol>

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The authors have no financial disclosures.

在这篇文章中，我们描述了miRNA表达一个简化的工作流程分析在存档FFPE和新鲜冷冻前列腺癌的临床组织。在前列腺癌，一些研究表明，在前列腺癌的发生，发展和转移的microRNA中起重要作用。然而，矛盾的结果往往是获得一个特定的miRNA 22以来的miRNA的提取和分析方法有很大的出入。鉴于越来越多的证据支持的miRNA作为替代的前列腺癌生物标志物的前列腺癌的预后，诊断和治疗的潜在的应用，有必要在临床组织中精确定量的miRNA表达谱。 我们采用定量实时PCR和原位杂交的组合为miRNA的分析的前列腺癌的临床标本。在这个流程中，我们优化了现有方法的miRNA的提取，表达Taq酶分析基于RT-PCR和ISH人引以LNA探针分析。在整个工作流，这是极其重要的，以保持一个无RNA酶的环境。使用的所有试剂/解决方案应无RNA酶和额外应谨慎处理RNA和其他试剂使用。所有实时反应，应设置在冰上，以尽量减少RNA降解。 在前列腺癌组织中表达准确分析往往是由异质性和前列腺癌病变25的多焦性阻碍。这可以通过使用一个激光捕获microdisssection技术允许的良性和恶性上皮细胞的分离，也减少了污染的基质，从而使前列腺组织22,25的准确的下游分子分析来克服。我们聘请的存档FFPE组织，然后通过miRNA的表达使用已报告是可靠的Taqman微小RNA表达分析分析LCM。 LCM组织的miRNA表达谱已signif着性可能提高miRNA的生物标志物的发现。事实上，在分析FFPE组织考虑到这些组织被广泛使用的病理学家进行临床分析，保存，归档和相当大的兴趣都一应俱全3。另外，由于其小尺寸及耐内源性RNase活性，miRNA是相对较少受FFPE依赖性降解和有吸引力潜在生物标志物3。福尔马林固定的组织的miRNA的表达谱已报道被紧密相关的那些冷冻组织21。 我们采用的和比较了miRNA的提取从FFPE组织中两种不同的商业试剂盒。同时兼具试剂盒，得到高品质的RNA用于RT-PCR分析，我们使用它使用一个简单，精简协议miRNA的一致和纯化总RNA从FFPE组织中之一。 诚信，纯度和RNA的建议立即进行删除的浓度前RT-PCR分析ð进行测试。样品显示出低A260 / A230之比（&lt;1.8）应该使用在RT反应之前重新沉淀并重新分析的纯度。此外，实时PCR临床组织的分析，可能需要进一步优化。阈值周期（Ct值）&gt; 33显示低的放大反映低起点cDNA模板。在这种情况下，cDNA模板输入可以增加。此外，它是最重要的是充分控制用于归以及确定正常和肿瘤组织之间的miRNA相对量词。内源性对照（RNU48 / RNU24 / RNU6B）基于扩增信号的正常的均匀性与肿瘤样品选择。 此外，我们描述了一种优化的协议为miRNA的ISH分析的基础上LNA-基于探针前列腺癌组织。我们已优化的miRNA ISH方法可应用于前列腺癌组织切片或前列腺癌组织微阵列（TMA），后者提供强效IAL加快miRNA的生物标志物的发现。该协议也可以被应用到其它类型的组织。然而，proteinaseKdigestionandparaformaldehydetreatment的持续时间，可能需要进行优化。蛋白酶K消化允许所述miRNA探针进入细胞，而多聚甲醛固定是需要防止下列透化的miRNA损失。该协议，最初是用于开发的LNA探头26进行了优化，得到一个特定的信号，在前列腺组织中最小的背景。探针浓度和温育条件为前列腺癌组织进行了优化。我们聘请5&#39;digoxigeninlabeledprobes在20-50纳米根据miRNA的丰度的浓度。然而，3&#39;digoxigeninlabeledprobes和双标记（5&#39;和3&#39;）的标记探针可被取代。事实上，双标记探针提供了可能用于小分子RNA检测色度更好的发展，并建议用于低丰度miRNA检测。另外，阻挡和抗体孵育持续时间可以被优化。较长的阻塞，建议降低的背景下，尤其是低丰度的miRNA。与BM紫色基板Thedurationofcolor反应，应密切监测。长时间孵育能够产生的背景染色。通常情况下，丰富的miRNA需要更短的孵育（1-2小时），而低丰度的miRNA与更长的孵化检测。期间ISH，它确保了组织切片不干燥过程中的任何步骤，因为这可能会导致染色的工件是重要的。 总之，虽然一些研究表明miRNA的重要前列腺癌生物标志物，几个这些研究的意义往往因相互矛盾的结果争论不休。在这里，我们已定义了精确的miRNA表达的优化工作流程中具有潜在加速miRNA的发现作为生物标志物用于前列腺癌前列腺癌的临床标本的分析。虽然此工作流有显著潜力在前列腺癌组织中的临床准确信息的miRNA表达，也有使用的技术固有的限制。低丰度的miRNA可能难以与优化miRNA的RT-PCR方法来分析和原位杂交分析描述。虽然基于RT-PCR分析具有更宽的动态范围比ISH 27,28，从LCM得到的低RNA的量可能会限制检测低丰度的miRNA。 ISH基于检测miRNA的产生强大的空间信息，但是其局限性在于它是一种半定量技术借助较低动态范围28。这种技术依赖于探针灵敏度采用的和特异性和对已进行杂交和检测的miRNA的条件的优化。

前列腺癌是一种常见的诊断男性恶性肿瘤即癌症相关死亡的男子居于领先地位的原因之一。在美国，估计有220800例新病例和27540例死亡将在2015年1报告。 前列腺癌是一种异质性疾病与高度可变的疾病课程 - 肿瘤可好逸恶劳或非常积极。在前列腺癌的临床管理的一个重要挑战是目前常用的方法/生物标志物的疾病筛查，诊断，预后和治疗2的不足所造成。当前的筛选方法包括前列腺特异性抗原（PSA）测试和直肠指检（DRE），随后前列腺活检3。前列腺特异性抗原（PSA）是使用最广泛的前列腺癌生物标志物已显著革命性临床管理和改进的存活率4。然而，由于粘合剂包括紫胶固有的局限性k的特异性，PSA为基础的筛选，导致过度的诊断和治疗上的疾病。鉴于此，积极努力正在指向一个搜索备用前列腺癌生物标志物，特别是那些可以预测疾病的攻击性和实现更好的治疗决策4,5。在过去的几年里，微RNA（miRNA）已成为有前途的替代前列腺癌生物标志物。 微RNA（miRNA）构成一个进化上保守类小的非编码RNA，它通过与同源的mRNA靶的3&#39;-非翻译区（UTR）序列特异性相互作用的基因表达抑制转录后。据估计，&gt;的mRNA的60％是保守的miRNA 6的目标。 miRNA基因位于基因间隔区或内含子或蛋白/非蛋白质的外显子编码基因7内。这些基因被RNA聚合酶Ⅱ优先转录成初步RY的miRNA（PRI-miRNA的，几千个碱基长）的形式发夹状茎环二级结构。这些PRI-miRNA的加工成miRNA前体（的pre-miRNA，60-75核苷酸长），其出口到细胞质和进一步加工成成熟miRNA（18-25核苷酸长）8-10。的miRNA调节关键的细胞过程，包括增殖，发育，分化和凋亡11。研究表明miRNA的表达谱在各种人类恶性肿瘤包括前列腺癌12-15普遍失调。 miRNA的表达谱已报道被广泛失调在原发性和转移性前列腺癌。改变的miRNA表达已与前列腺癌的进展，攻击性和复发突出的miRNA 12,14,16-19的预后潜力。越来越多的证据体表明miRNA的前列腺癌症的发生，发展，进步起到重要的作用机理离子和转移。总体而言，微RNA正在成为有前途的替代生物标志物前列腺癌的诊断和预后，可在前列腺肿瘤12分层正常，癌组织和援助加以区分。此外，miRNA是发展的有效的治疗方法预防前列腺癌20的重要目标。 由于它们的小尺寸和抗内源性RNase活性，miRNA是，可以在福尔马林固定的组织21和前列腺活检22容易地检测的生物标志物的稳定。此外，miRNA的表达谱进行了比较在冷冻和福尔马林固定的组织，并已被发现是强烈相关21。然而，miRNA表达谱的前列腺癌临床组织往往是具有挑战性的，由于肿瘤的异质性，抽样误差，间质污染等miRNA的有效生物标记物的前列腺癌严重RELI的发展ES它们在临床组织中精确检测。在这里，我们描述了在我们的实验室进行miRNA表达谱的存档FFPE和新鲜冷冻前列腺癌的临床标本使用简化的工作流程。我们使用定量实时PCR的组合和在对miRNA的原位杂交分析临床标本的，前者得到更多量化信息，后者用于可视化潜在的miRNA标志物的差异表达的组织中的阵列。在这个工作流程中，我们优化的miRNA提取从FFPE和冷冻前列腺组织的现有方法，表达由基于miRNA的RT-PCR和miRNA的原位杂交技术利用锁核酸（LNA）的Taqman探针分析为基础的探针23。 LNA基于探头提供增加的灵敏度和特异性相比DNA-或RNA-基于探针和使所有miRNA序列的鲁棒检测，无论其GC含量也允许discrimina化miRNA家族的。我们已优化的miRNA ISH方法可应用于前列腺癌组织切片或前列腺癌组织微阵列（TMA），后者提供到加速miRNA的生物标志物发现的潜力。

<table><tbody><tr><td>Microtome</td><td>Leica Biosystems&amp;nbsp;</td><td>RM2255</td><td></td></tr><tr><td>Arcturus Autopix for LCM</td><td>Arcturus/ Life Technologies</td><td>LCM1621/LCM1110</td><td>Alternatively, Arcutus Xt system from Life Technolgies can be used.&amp;nbsp;</td></tr><tr><td>CapSure Macro LCM Caps</td><td>Life Technologies</td><td>LCM0211</td><td></td></tr><tr><td>miRNeasy FFPE Kit&amp;nbsp;</td><td>Qiagen</td><td>217504</td><td></td></tr><tr><td>7500 Fast Real-time PCR System&amp;nbsp;</td><td>Applied Biosystems/ Life Technologies</td><td>4351106</td><td></td></tr><tr><td>Taqman MicroRNA Reverse Transcription kit&amp;nbsp;</td><td>Applied Biosystems/ Life Technologies</td><td>4366596</td><td></td></tr><tr><td>Taqman Fast Universal PCR master mix&amp;nbsp;</td><td>Applied Biosystems/ Life Technologies</td><td>4352042</td><td></td></tr><tr><td>DIG labeled LNA probe for U6</td><td>Exiqon</td><td>99002-01</td><td></td></tr><tr><td>BM Purple AP substrate</td><td>Roche</td><td>11442074001</td><td></td></tr><tr><td>Pre-hybridization solution&amp;nbsp;</td><td>Biochain</td><td>&lt;a href=&quot;http://www.biochain.com/coa/K21910XXCA.pdf&quot;&gt;K2191050-1&lt;/a&gt;</td><td></td></tr><tr><td>Hybridization solution&amp;nbsp;</td><td>Biochain</td><td>&lt;a href=&quot;http://www.biochain.com/coa/K21910XXCA.pdf&quot;&gt;K2191050-2&lt;/a&gt;</td><td></td></tr><tr><td>Blocking solution&amp;nbsp;</td><td>Biochain</td><td>&lt;a href=&quot;http://www.biochain.com/coa/K21910XXCA.pdf&quot;&gt;K2191050-8&lt;/a&gt;</td><td></td></tr><tr><td>AP-conjugated anti-digoxigenin antibody</td><td>Biochain</td><td>&lt;a href=&quot;http://www.biochain.com/coa/K21910XXCA.pdf&quot;&gt;K2191050-7&lt;/a&gt;</td><td></td></tr><tr><td>Aqueous mounting media&amp;nbsp;</td><td>Vector Laboratories&amp;nbsp;</td><td>H-5501&amp;nbsp;&amp;nbsp;</td><td></td></tr><tr><td>Trizol (guanidine isothiocyanate-phenol reagent)&amp;nbsp;</td><td>Life Technologies</td><td>&lt;a href=&quot;https://www.lifetechnologies.com/order/catalog/product/15596018&quot;&gt;15596-018&lt;/a&gt;</td><td></td></tr><tr><td>Harris hematoxylin</td><td>Statlab</td><td>SL200</td><td></td></tr><tr><td>Eosin&amp;nbsp;</td><td>Statlab</td><td>SL201&amp;nbsp;</td><td></td></tr></tbody></table>

mirna expression analyses in prostate cancer clinical tissues

在前列腺癌（PCA）的临床管理的一个重要挑战是目前使用的生物标志物的疾病筛查，诊断，预后和治疗的不足所造成。近年来，微RNA（miRNA）已成为有前途的替代标志物前列腺癌的诊断和预后。然而，miRNA的有效生物标记前列腺癌的发展在很大程度上依赖于他们在临床组织中准确的检测。 miRNA的分析了前列腺癌的临床标本往往是具有挑战性的，由于肿瘤的异质性，抽样误差，间质污染等。这篇文章的目的是描述一个简化的工作流程在存档FFPE和新鲜冷冻前列腺癌的临床标本的miRNA分析使用的组合定量实时PCR（RT-PCR），原位杂交（ISH）。在这个流程中，我们优化了miRNA的提取从FFPE现有的方法和冷冻前列腺Tissu酒店ES和表达分析了基于miRNA的RT-PCR的Taqman探针。此外，我们描述了ISH优化方法分析了使用锁核酸（LNA），前列腺组织formiRNA探测 - 基于探针。我们已优化的miRNA ISH方法可应用于前列腺癌组织切片或前列腺癌组织微阵列（TMA）。

的miR-203的LCM原发性前列腺癌临床标本表达谱，通过RT-PCR分析（图1） RT-PCR分析相对的miR-203表达在LCM原发性前列腺癌组织和匹配的邻近的正常区域进行与在赛尼等人 15 RNU48所述的用作对照。下面的表总结了在前列腺癌肿瘤组织相对于邻近的正常组织的相对的miR-203的表达。 在显微的miR-383的表达通过RT-PCR比较分析VS激光捕获显微切割的前列腺组织（图2） 前列腺癌组织中或者在显微镜下显微切割或经受LCM随后通过RT-PCR在肿瘤组织分析的miR-383的表达相对于在肿瘤样品中旁正常tissues.Relative的miR-383的表达被shown.RNU48被用作对照。 如图2，观察到的差异时的miRNA表达在这些组织中进行分析。 LCM拥有显微切割的优点显微镜下，因为它允许前列腺组织的相对纯的人口和隔离更加可靠。 ISH在前列腺癌的临床组织中分析的miR-203的表达（图3） 原位杂交在正常和骨转移性前列腺癌组织的miR-203表达的分析物分析用5&#39;-DIG标记的特异于的miR-203和LNA探针进行如赛尼等人的15前列腺癌组织微阵列被用于ISH U6。 ISH的代表性的例子分析示于图3中，图中从赛尼等人部分地再现 。（2011）15图 3A和3B showsmiR-203 EXPRES锡永（左）和在指定的组织中的U6控制表达（右）。 ISH信号进行半定量地基于染色和染色的细胞的百分比的强度等级，并分配1〜4强度的miR-203表达的分数得分的那些U6表达被划分，以获得归一化的miR-203的表达水平是被绘制在图3C中 。 <img alt="图1" src="/files/ftp_upload/53123/53123fig1.jpg" /> 图1:的miR-203的LCM原发性前列腺癌临床标本表达谱，通过RT-PCR分析 。在LCM-前列腺癌组织和匹配的邻近正常区域相对的miR-203表达的RT-PCR分析。 RNU48被用作对照。下表总结了前列腺癌肿瘤组织相对于邻近正常组织的相对的miR-203表达。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53123/53123fig1large.jpg" target="_blank">请点击这里</a>查看该图的放大版本。 <img alt="图2" src="/files/ftp_upload/53123/53123fig2.jpg" /> 图2:的miR-383的表达通过RT-PCR比较在显微分析与激光捕获显微切割的前列腺癌组织中的前列腺癌组织中或者在显微镜下显微切割或经受LCM随后通过RT-PCR分析中的miR-383 的表达。肿瘤组织相匹配的邻近正常组织。肿瘤样本中相对的miR-383表达的shown.RNU48作为对照。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53123/53123fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图3" src="/files/ftp_upload/53123/53123fig3.jpg" /> 图3:ISH分析了前列腺癌的临床组织中的miR-203表达的这一数字已经被修改15。组织进行杂交DIG-拉贝如在15中所述 LLED锁核酸（LNA），基于探针的mir-203和U6（对照）。的miR-203表达（左图）和对照U6表达（右图）在人类前列腺癌细胞和正常骨转移组织的ISH分析的代表性例子，显示衰减骨转移的组织中的miR-203表达。在更高的放大倍数（40倍）示于代表ISH例子。在正常前列腺组织和骨转移性前列腺癌组织中相对的miR-203表达水平的评估，ISH分析。的miR-203表达得分，U6标准化的分数，并绘制。水平线表示平均值。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53123/53123fig3large.jpg" target="_blank">请点击此处查看该图的放大版本。</a>

Watch this Scientific Journal Video about miRNA Expression Analyses in Prostate Cancer Clinical Tissues at JoVE.com

miRNA Expression Analyses in Prostate Cancer Clinical Tissues

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, ...

mirna-expression-analyses-in-prostate-cancer-clinical-tissues

Nanyang Technological University

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JoVE Journal

Medicine

10.8K 次观看.  University of California San Francisco. 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).

视频: miRNA Expression Analyses in Prostate Cancer Clinical Tissues

MicroRNA Detection in Prostate Tumors by Quantitative Real-time PCR (qPCR)

MicroRNAs (miRNAs) are single-stranded, 18&#x2013;24 nucleotide long, non-coding RNA molecules. They are involved in virtually every cellular process including development1, apoptosis2, and cell cycle regulation3. MiRNAs are estimated to regulate the expression of 30% to 90% of human genes4 by binding to their target messenger RNAs (mRNAs)5. Widespread dysregulation of miRNAs has been reported in various diseases and cancer subtypes6. Due to their prevalence and unique structure, these small molecules are likely to be the next generation of biomarkers, therapeutic agents and/or targets.
Methods used to investigate miRNA expression include SYBR green I dye- based as well as Taqman-probe based qPCR. If miRNAs are to be effectively used in the clinical setting, it is imperative that their detection in fresh and/or archived clinical samples be accurate, reproducible, and specific. qPCR has been widely used for validating expression of miRNAs in whole genome analyses such as microarray studies7. The samples used in this protocol were from patients who underwent radical prostatectomy for clinically localized prostate cancer; however other tissues and cell lines can be substituted in. Prostate specimens were snap-frozen in liquid nitrogen after resection. Clinical variables and follow-up information for each patient were collected for subsequent analysis8.
Quantification of miRNA levels in prostate tumor samples. The main steps in qPCR analysis of tumors are: Total RNA extraction, cDNA synthesis, and detection of qPCR products using miRNA-specific primers. Total RNA, which includes mRNA, miRNA, and other small RNAs were extracted from specimens using TRIzol reagent. Qiagen's miScript System was used to synthesize cDNA and perform qPCR (Figure 1). Endogenous miRNAs are not polyadenylated, therefore during the reverse transcription process, a poly(A) polymerase polyadenylates the miRNA. The miRNA is used as a template to synthesize cDNA using oligo-dT and Reverse Transcriptase. A universal tag sequence on the 5' end of oligo-dT primers facilitates the amplification of cDNA in the PCR step. PCR product amplification is detected by the level of fluorescence emitted by SYBR Green, a dye which intercalates into double stranded DNA. Specific miRNA primers, along with a Universal Primer that binds to the universal tag sequence will amplify specific miRNA sequences. 
The miScript Primer Assays are available for over a thousand human-specific miRNAs, and hundreds of murine-specific miRNAs. Relative quantification method was used here to quantify the expression of miRNAs. To correct for variability amongst different samples, expression levels of a target miRNA is normalized to the expression levels of a reference gene. The choice of a gene on which to normalize the expression of targets is critical in relative quantification method of analysis. Examples of reference genes typically used in this capacity are the small RNAs RNU6B, RNU44, and RNU48 as they are considered to be stably expressed across most samples. In this protocol, RNU6B is used as the reference gene.

MicroRNA Detection in Prostate Tumors by Quantitative Real-time PCR qPCR

Quantitative Real Time polymerase chain reaction (qPCR) is a rapid and sensitive method to investigate the expression levels of various microRNA (miRNA) molecules in tumor samples. Using this method expression of hundreds of different miRNA molecules can be amplified, quantified, and analyzed from the same cDNA template.

实时定量PCR（qPCR的microRNA检测在前列腺肿瘤）

MicroRNAs (miRNAs) are single-stranded, 18&#x2013;24 nucleotide long, non-coding RNA molecules. They are involved in virtually every cellular process ...

科研

医学

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

Microarray-based Identification of Individual HERV Loci Expression: Application to Biomarker Discovery in Prostate Cancer

The prostate-specific antigen (PSA) is the main diagnostic biomarker for prostate cancer in clinical use, but it lacks specificity and sensitivity, particularly in low dosage values1&#8203;&#8203;. &#8216;How to use PSA&#39; remains a current issue, either for diagnosis as a gray zone corresponding to a concentration in serum of 2.5-10 ng/ml which does not allow a clear differentiation to be made between cancer and noncancer2 or for patient follow-up as analysis of post-operative PSA kinetic parameters can pose considerable challenges for their practical application3,4. Alternatively, noncoding RNAs (ncRNAs) are emerging as key molecules in human cancer, with the potential to serve as novel markers of disease, e.g. PCA3 in prostate cancer5,6 and to reveal uncharacterized aspects of tumor biology. Moreover, data from the ENCODE project published in 2012 showed that different RNA types cover about 62% of the genome. It also appears that the amount of transcriptional regulatory motifs is at least 4.5x higher than the one corresponding to protein-coding exons. Thus, long terminal repeats (LTRs) of human endogenous retroviruses (HERVs) constitute a wide range of putative/candidate transcriptional regulatory sequences, as it is their primary function in infectious retroviruses. HERVs, which are spread throughout the human genome, originate from ancestral and independent infections within the germ line, followed by copy-paste propagation processes and leading to multicopy families occupying 8% of the human genome (note that exons span 2% of our genome). Some HERV loci still express proteins that have been associated with several pathologies including cancer7-10. We have designed a high-density microarray, in Affymetrix format, aiming to optimally characterize individual HERV loci expression, in order to better understand whether they can be active, if they drive ncRNA transcription or modulate coding gene expression. This tool has been applied in the prostate cancer field (Figure 1).

Human endogenous retroviruses (HERV), which occupy 8% of the human genome, retain scarce coding capacities but a hundred thousand long terminal repeats (LTRs). A custom Affymetrix microarray was designed to identify individual HERV locus expression and was used on prostate cancer tissues as a proof of concept for future clinical studies.

应用生物标志物发现在前列腺癌：个别赫尔维位点表达微阵列鉴定

The prostate-specific antigen (PSA) is the main diagnostic biomarker for prostate cancer in clinical use, but it lacks specificity and sensitivity, ...

An Orthotopic Murine Model of Human Prostate Cancer Metastasis

Our laboratory has developed a novel orthotopic implantation model of human prostate cancer (PCa). As PCa death is not due to the primary tumor, but rather the formation of distinct metastasis, the ability to effectively model this progression pre-clinically is of high value. In this model, cells are directly implanted into the ventral lobe of the prostate in Balb/c athymic mice, and allowed to progress for 4-6 weeks. At experiment termination, several distinct endpoints can be measured, such as size and molecular characterization of the primary tumor, the presence and quantification of circulating tumor cells in the blood and bone marrow, and formation of metastasis to the lung. In addition to a variety of endpoints, this model provides a picture of a cells ability to invade and escape the primary organ, enter and survive in the circulatory system, and implant and grow in a secondary site. This model has been used effectively to measure metastatic response to both changes in protein expression as well as to response to small molecule therapeutics, in a short turnaround time.

This orthotopic model of human prostate cancer allows for quantification of tumor size, circulating tumor cells, and formation of distinct metastasis to the lung. As cells must escape the primary organ, enter the blood stream, and implant into a secondary site, this model effectively recapitulates the scenario in humans.

人类前列腺癌转移的原位小鼠模型

Our laboratory has developed a novel orthotopic implantation model of  human prostate cancer (PCa). As PCa death is not due to the primary tumor, but  ...

Profiling of Estrogen-regulated MicroRNAs in Breast Cancer Cells

Estrogen plays vital roles in mammary gland development and breast cancer progression. It mediates its function by binding to and activating the estrogen receptors (ERs), ER&#945;, and ER&#946;. ER&#945; is frequently upregulated in breast cancer and drives the proliferation of breast cancer cells. The ERs function as transcription factors and regulate gene expression. Whereas ER&#945;&#39;s regulation of protein-coding genes is well established, its regulation of noncoding microRNA (miRNA) is less explored. miRNAs play a major role in the post-transcriptional regulation of genes, inhibiting their translation or degrading their mRNA. miRNAs can function as oncogenes or tumor suppressors&#160;and are also promising biomarkers. Among the miRNA assays available, microarray and quantitative real-time polymerase chain reaction (qPCR) have been extensively used to detect and quantify miRNA levels. To identify miRNAs regulated by estrogen signaling in breast cancer, their expression in ER&#945;-positive breast cancer cell lines were compared before and after estrogen-activation using both the &#181;Paraflo-microfluidic microarrays and Dual Labeled Probes-low density arrays. Results were validated using specific qPCR assays, applying both Cyanine dye-based and Dual Labeled Probes-based chemistry. Furthermore, a time-point assay was used to identify regulations over time. Advantages of the miRNA assay approach used in this study is that it enables a fast screening of mature miRNA regulations in numerous samples, even with limited sample amounts. The layout, including the specific conditions for cell culture and estrogen treatment, biological and technical replicates, and large-scale screening followed by in-depth confirmations using separate techniques, ensures a robust detection of miRNA regulations,&#160;and eliminates false positives and other artifacts. However, mutated or unknown miRNAs, or regulations at the primary and precursor transcript level, will not be detected. The method presented here represents a thorough investigation of estrogen-mediated miRNA regulation.

Molecular signaling through both estrogen and microRNAs are critical in breast cancer development and growth. Estrogen activates the estrogen receptors, which are transcription factors. Many transcription factors can regulate the expression of microRNAs, and estrogen-regulated microRNAs can be profiled using different large-scale techniques.

在乳腺癌细胞雌激素调节小分子RNA的剖析

Estrogen plays vital roles in mammary gland development and breast cancer progression. It mediates its function by binding to and activating the estrogen ...

TMEM200A：一种在胃癌中调节 EMT 和 PI3K/AKT 的新型癌基因

Multiomics Analysis of TMEM200A as a Pan-Cancer Biomarker

The transmembrane protein, TMEM200A, is known to be associated with human cancers and immune infiltration. Here, we assessed the function of TMEM200A in common cancers by multiomics analysis and used in vitro cell cultures of gastric cells to verify the results. The expression of TMEM200A in several human cancer types was assessed using the RNA-seq data from the UCSC Xena database. Bioinformatic analysis revealed a potential role of TMEM200A as a diagnostic and prognostic biomarker. 
Cultures of normal gastric and cancer cell lines were grown and TMEM200A was knocked down. The expression levels of TMEM200A were measured by using quantitative real-time polymerase chain reaction and western blotting. In vitro loss-of-function studies were then used to determine the roles of TMEM200A in the malignant behavior and tumor formation of gastric cancer (GC) cells. Western blots were used to assess the effect of the knockdown on epithelial-mesenchymal transition (EMT) and PI3K/AKT signaling pathway in GC. Bioinformatic analysis showed that TMEM200A was expressed at high levels in GC. 
The proliferation of GC cells was inhibited by TMEM200A knockdown, which also decreased vimentin, N-cadherin, and Snai proteins, and inhibited AKT phosphorylation. The PI3K/AKT signaling pathway also appeared to be involved in TMEM200A-mediated regulation of GC development. The results presented here suggest that TMEM200A regulates the tumor microenvironment by affecting the EMT. TMEM200A may also affect EMT through PI3K/AKT signaling, thus influencing the tumor microenvironment. Therefore, in pan-cancers, especially GC, TMEM200A may be a potential biomarker and oncogene.

作者聚焦：揭示胃癌中跨膜蛋白家族相关标志物及其对靶向治疗的影响 

Here, a protocol is presented in which multiple bioinformatic tools are combined to study the biological functions of TMEM200A in cancer. In addition, we also experimentally validate the bioinformatics predictions.

TMEM200A作为泛癌生物标志物的多组学分析

The transmembrane protein, TMEM200A, is known to be associated with human cancers and immune infiltration. Here, we assessed the function of TMEM200A in ...

Optimization of a Multiplex RNA-based Expression Assay Using Breast Cancer Archival Material

Nucleic acid degradation in archival tissue, tumor heterogeneity, and a lack of fresh frozen tissue specimens can negatively impact cancer diagnostic services in pathology laboratories worldwide. Gene amplification and expression diagnostic testing using archival material or material that requires transportation to servicing laboratories, needs a more robust and accurate test adapted to current clinical workflows. Our research team optimized the use of Invitrogen&#8482; QuantiGene&#8482; Plex Assay (Thermo Fisher Scientific) to quantify RNA in archival material using branched-DNA (bDNA) technology on Luminex xMAP&#174; magnetic beads. The gene expression assay described in this manuscript is a novel, quick, and multiplex method that can accurately classify breast cancer into the different molecular subtypes, omitting the subjectivity of interpretation inherent in imaging techniques. In addition, due to the low input of material required, heterogeneous tumors can be laser microdissected using Hematoxylin and Eosin (H&#38;E) stained sections. This method has a wide range of possible applications including tumor classification with diagnostic potential and measurement of biomarkers in liquid biopsies, which would allow better patient management and disease monitoring. In addition, the quantitative measurement of biomarkers in archival material is useful in oncology research with access to libraries of clinically-annotated material, in which retrospective studies can validate potential biomarkers and their clinical outcome correlation.

Nucleic acid degradation in archival tissue, tumor heterogeneity, and a lack of fresh frozen tissue specimens can negatively impact cancer diagnostic services in pathology laboratories worldwide. This manuscript describes the optimization of a panel of biomarkers using a multiplex magnetic bead assay to classify breast cancers.

应用乳腺癌档案材料的多路 RNA 表达方法的优化

Nucleic acid degradation in archival tissue, tumor heterogeneity, and a lack of fresh frozen tissue specimens can negatively impact cancer diagnostic ...

生物工程

Laser-capture Microdissection of Human Prostatic Epithelium for RNA Analysis

The prostate gland contains a heterogeneous milieu of stromal, epithelial, neuroendocrine and immune cell types. Healthy prostate is comprised of fibromuscular stroma surrounding discrete epithelial-lined secretory lumens and a very small population of immune and neuroendocrine cells. In contrast, areas of prostate cancer have increased dysplastic luminal epithelium with greatly reduced or absent stromal population. Given the profound difference between stromal and epithelial cell types, it is imperative to separate the cell types for any type of downstream molecular analysis. Despite this knowledge, the bulk of gene expression studies compare benign prostate to cancer without micro-dissection, leading to stromal bias in the benign samples. Laser-capture micro-dissection (LCM) is an effective method to physically separate different cell types from a specimen section. The goal of this protocol is to show that RNA can be successfully isolated from LCM-collected human prostatic epithelium and used for downstream gene expression studies such as RT-qPCR and RNAseq.

The goal of this protocol is to use laser-capture micro-dissection as an effective method to isolate pure populations of cell types from heterogeneous prostate tissues for downstream RNA analysis.

在前列腺癌的临床组织中miRNA的表达分析

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此视频中的章节

实时定量PCR（qPCR的microRNA检测在前列腺肿瘤）

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

应用生物标志物发现在前列腺癌：个别赫尔维位点表达微阵列鉴定

人类前列腺癌转移的原位小鼠模型

在乳腺癌细胞雌激素调节小分子RNA的剖析

TMEM200A作为泛癌生物标志物的多组学分析

TMEM200A作为泛癌生物标志物的多组学分析

TMEM200A作为泛癌生物标志物的多组学分析

应用乳腺癌档案材料的多路 RNA 表达方法的优化

人类前列腺上皮的激光捕获显微切割的RNA分析