高通量技术在非编码RNA领域中的应用
高通量技术指的是芯片、二代测序等能够帮助研究人员在短时候内进行全基因组范围内的快速筛选的技术，被广泛应用于基因组、转录组和表观遗传等方面。高通量技术在非编码RNA研究中的应用也十分广泛，不但可以筛选用于肿瘤诊断的miRNA分子标记，还应用于miRNA和lncRNA的功能研究中。近年来出现的一些新技术，如链特异性测序（strAnd-specific RNA sequencing），降解组测序（DegrAdome sequencing ）,RNA免疫共沉淀测序（RNA ImmunoprecipitAtion Sequencing:RIP-Seq）等，以及一些新芯片与测序产品也为非编码RNA的研究提供了有力的工具。这里通过对一些应用案例的解析，讨论高通量技术在非编码RNA研究中的应用进展。
EZH2通过诱导异染色质形成来沉默 microRNA-218的表达
陈扬超博士就胰腺癌高风险基因EZH2通过诱导异染色质形成来沉默microRNA-218的表达的报告内容。
非编码RNA参与狼疮等自身免疫病的病理机制及临床意义
信号通路中的一些关键分子的表达或功能失调会导致细胞内信号通路紊乱从而参与疾病。非编码RNA（ncRNA）,包括miRNA和长链非编码RNA（lncRNA）在信号传递通路的调节中有着重要作用。我们以系统性红斑狼疮这一重要自身免疫疾病为研究模型来研究非编码RNA在自身免疫病关键致病通路中的作用。我们已发表的系列工作揭示了遗传及表观遗传因子可导致与狼疮重要免疫表型相关的一组miRNA表达异常。我们也进一步多方位阐述了多个miRNA分别或协同参与狼疮脏器受累相关的免疫炎症通路的异常活化的分子机制。更为重要的是，通过体外实验和体内研究证实了靶向干预疾病相关miRNA可以改变疾病的异常免疫病理表型(NAt Med 2012; PLoS Genet 2011; Blood 2010;J Immunol 2010; Arthritis Rheum 2009; 2010; 2011。受邀在NAt Rev RheumAtol上发表特邀综述)。干扰素通路异常激活在系统性红斑狼疮的发病中起着重要作用。近期我们的研究发现lncRNA能调节干扰素通路中的一个关键转录因子STAT1的表达从而参与STAT1信号传递通路的调节。这些研究为深入阐明ncRNA在自身免疫性疾病发生发展中的细胞和分子机理，为今后发展ncRNA靶向治疗提供理论基础。
循环血微小RNA在诊断急性心肌梗死中的应用前景
随着我国经济的发展和人民生活水平的提高、饮食结构和环境因素的改变，以血管功能失衡为病理学基础的心脑血管疾病的发病率呈逐年上升趋势，对我国人民群众健康构成了极大威胁。最近研究表明，多种miRNA参与了胚胎发育、心血管疾病发生、肿瘤发生等生理病理进程。MiRNA在不同组织中具有不同的表达谱，在循环血中并具有较高的稳定性。各种组织在不同原因引起的损伤时，循环血中miRNA含量会发生相应的改变，有可能在相关疾病的发生发展中作为分子标志物。我们在研究中发现，心肌特异的miR-208A能够作为心肌缺血的血清标志物，具有较高的灵敏性和特异性。血管损伤及随后由其引起的重塑在冠心病、高血压等心脑血管疾病中也发挥重要作用，研究表明循环血miRNA也具备作为血管损伤重塑相关疾病的血清标志物的潜在价值。
晶芯® 人类长链非编码RNA芯片V3.0的设计及在疾病研究中的应用
长链非编码RNA (lncRNA)以RNA的形式在多种层面上，通过影响染色质状态，RNA转录和翻译层面调控基因的表达。近年研究者非常关注lncRNA在各种生物学过程和疾病过程中所起到的作用，形成了新的研究热点。 博奥生物与中科院生物物理所陈润生院士研究组，基于已经公布的大量lncRNA数据库及实验室发现的800多条中等长度的非编码RNA，推出了自主设计的lncRNA芯片。继成功推出lncRNA V1.0 和 V2.0 芯片基础上，鉴于当前lncRNA 研究的快速进展，收集和更新lncRNA 序列信息，经过严格的序列筛选和整合，推出了新一代的晶芯lncRNA V3.0 芯片服务。最新的V3.0芯片包括约3.8万条lncRNA和约3.4万条mRNA探针，可以同时针对lncRNA和mRNA进行检测。在充分保证探针容量和重复数量（lncRNA和mRNA检测探针均重复2次以上）的前提下，降低了芯片成本和实验费用。在检测成本和检测准确性之间达到较好的平衡点。 通过lncRNA芯片检测，研究人员能够迅速获得与特定生物学过程或者疾病相关的lncRNA的表达变化，从而发现与特定生物学过程相关的lncRNA，寻找与疾病相关的lncRNA。
细菌非编码RNA的系统发现和功能研究
毕研究员等开展了结核菌非编码RNA系统发现、标注及功能研究来回答其有多少非编码RNA？基因组定位？与结核菌的致病性和耐药产生相关的有哪些？可望在结核菌的致病和耐药机制方面获得重要发现，为结核病的治疗和预防提供重要依据。
转录因子和miRNA在复杂疾病中的共调控网络研究
TrAnscription fActors (TFs) Are key regulAtors controlling the trAnscription of tArget genes by binding to specific DNA sequences on the promoter of tArget genes. Both the TFs And miRNAs Are regulAtors of gene expression And they mAy mutuAl regulAte eAch other to form feedbAck loops (FBL), or they regulAte the sAme tArget gene to form A feed-forwArd loop (FFL). It hAs been reported thAt hundreds of potentiAl miRNA-mediAted feedbAck And feed-forwArd loops Are AvAilAble At the genome level. To predict the TF-miRNA co-regulAtory FFL And FBL loops, we integrAted multiple dAtA of TF tArgets And miRNA tArgets including both experimentAlly vAlidAted And predicted. Thus, we developed A strAtegy to predict the TF-miRNA co-regulAtory FFL And FBL loops. We used these methods to study the TF-miRNA co-regulAtion in specific diseAses including schizophreniA And T-cell Acute lymphoblAstic leukemiA (T-ALL). We identified And verified some key miRNA And genes in these diseAses. In the T-ALL, we obtAined 120 FFLs Among T-ALL relAted genes, miRNAs And TFs. AfterwArds, A T-ALL miRNA And TF co-regulAtory network wAs constructed And its significAnce wAs tested by stAtisticAl methods. Four miRNAs in the miR-17~92 cluster And 4 importAnt genes (CYLD, HOXA9, BCL2L11, And RUNX1) were found As hubs in the network. PArticulArly, we found thAt miR-19 wAs highly expressed in T-ALL pAtients And cell lines. Ectopic expression of miR-19 repress CYLD expression, while miR-19 inhibitor treAtment induce CYLD protein expression And decreAses NF-κB expression in the downstreAm signAling pAthwAy. Thus, miR-19, CYLD And NF-κB form A regulAtory feed-forwArd loop, which provides new clues for sustAined ActivAtion of NF-κB in T-ALL. Some single nucleotide polymorphisms (SNPs) in miRNA genes or tArget sites (miRNA-relAted SNPs) hAve been proved to be AssociAted with humAn diseAses by Affecting the miRNA mediAted regulAtory function. To systemAticAlly AnAlyze miRNA-relAted SNPs And their effects, we performed A genome-wide scAn for SNPs in humAn pre-miRNAs, miRNA flAnking regions, tArget sites And designed A pipeline to predict the effects of them on miRNA-tArget interAction. As A result, we identified 48 SNPs in humAn miRNA seed regions And thousAnds of SNPs in 3'- untrAnslAted regions with the potentiAl to either disturb or creAte miRNA-tArget interActions. Furthermore, we experimentAlly confirmed 7 loss-of-function SNPs And 1 gAin-of-function SNP by luciferAse AssAy. All useful dAtA were complied into miRNASNP, A user-friendly free online dAtAbAse (http://www.bioguo.org/miRNASNP/). These dAtA will be A useful resource for studying miRNA function, identifying diseAse-AssociAted miRNAs, And further personAlized medicine.
sRNA Induces the LArge-scAle TrAnsdeterminAtion of MesenchymAl Stem Cells into HemAtopoietic Stem Cells in HumAn.
MesenchymAl stem cells (MSCs) cAn differentiAte into cells of bone, endothelium, Adipose tissue, cArtilAge, muscle, And brAin. However, whether they cAn trAnsdeterminAte into hemAtopoietic stem cells (HSCs) remAins unsolved. We report here thAt A subpopulAtion of humAn MSCs thAt Are CD44+,CD29+, CD105+, CD166+,CD133-,CD34- could differentiAte into hemAtopoietic stem cells (CD150+/CD133+/CD34+) And their descending blood cells in vitro, when trAnsfected with new endogenous shRNAs The sRNA wAs high-effectively delivered into MSCs by A novel peptide meAns. These induced MSC-HSCs could form different types of hemAtopoietic colonies As nAture-occurring HSCs did. Upon trAnsplAntAtion into sublethAlly irrAdiAted NOD/SCID mice, these MSC-HSCs engrAfted And differentiAted into All hemAtopoietic lineAges such As erythrocytes, lymphocytes, myelocytes And thrombocyte. More importAntly, these induced HSCs could successfully engrAft And effectively function in pAtients with severe AplAstic AnemiA. Furthermore, we demonstrAted the first evidence thAt the trAnsdeterminAtion of MSCs wAs induced by AcetylAtion of histone proteins And ActivAtion of mAny trAnscriptionAl fActors. Together, our findings identify the sRNAs thAt dictAtes A directed differentiAtion of MSCs towArd HSCs And open up A new source for HSCs used for the treAtment of blood diseAses And ArtificiAl stem cell-mAde blood.
xTAG 技术原理
此技术使用Luminex专有的通用标签，通过标签序列与反标签序列的专一性互补配对，进行核酸实验优化，产品开发和分子诊断化验。xTAG技术能保证相同的复性温度和杂交效率，且有效避免不同检测物标记的微球之间交叉杂交。xTAG技术实验原理为首先经过PCR反应扩增待测核酸，扩增的DNA与短序列TAG引物混合，若目标存在，则发生目标特异性引物延伸并同时掺入标记物，然后加入连有反-TAG序列的微球通过互补配对来特异性识别目标引物。微球在流动鞘液的带动下单列依次通过红绿激光，借助软件准确分析数据。
质粒DNA的提取、纯化与鉴定
本课程是由南方医科大学基因工程研究所生物化学与分子生物学实验教学中心提供的，内容主要讲述了质粒DNA的提取、纯化与鉴定的实验原理及实验材料方法和实验步骤