circRNA研究技术手册文件下载

一、circRNA概述

circRNAs(Circular RNAs,HUANZHUANGRNAFENZI)SHIYILEIBUJUYOU5'MODUANMAOZIHE3'MODUANpoly(A)WEIBA、BINGYIGONGJIAJIANXINGCHENGHUANXINGJIEGOUDEFEIBIANMARNAFENZI。circRNASHIQUBIEYUCHUANTONGXIANXINGRNADEYILEIXINXINGRNA,JUYOUBIHEHUANZHUANGJIEGOU,DALIANGCUNZAIYUZHENHEZHUANLUZUZHONG。DABUFENDEHUANZHUANGRNA SHIYOUWAIXIANZIXULIEGOUCHENG,ZAIBUTONGDEWUZHONGZHONGJUYOUBAOSHOUXING,TONGSHICUNZAIZUZHIJIBUTONGFAYUJIEDUANDEBIAODATEYIXING。YOUYUHUANZHUANGRNADUIHESUANMEIBUMINGAN,SUOYIBIXIANXINGRNAGENGWEIWENDING,ZHEISHIDEHUANZHUANGRNAZAIZUOWEIXINXINGLINCHUANGZHENDUANBIAOJIWUDEKAIFAYINGYONGSHANGJUYOUMINGXIANYOUSHI。MUQIANDUIYUcircRNADEYANJIUHAIZHISHIBINGSHANYIJIAO,circRNAsDESHENGWUXUEGONGNENGYEYOUDAIWAJUE,YINCISHIFEICHANGZHIDESHENRUDEYANJIUFANGXIANG。

二、常规的circRNA研究思路如下图所示。

 

TU1 circRNACHANGGUIYANJIUSILU

 

 

 

1、circRNA的已知功能有如下3种:

1)DIAOJIEmiRNAHUOXING-ceRNAZUOYONGMOSHI;

2)DIAOJIEXUANZEXINGJIANJIE(alternative splicing)-YUpre-mRNAJINGZHENGJIANJIE;

3)DIAOJIERNAJIEHEDANBAI(RBPs)DECUNCHU,FENLEIHEDINGWEI-YUDANBAIZHIJIEJIEHE;

SHANGSHUSANZHONGcircRNAJIZHIZUOYONGFANGSHI,2)HE3)YOUYUXUYAOYONGDAORIPHUORNA pull-down,SHIYANZHIXINGNANDUDAFENGXIANGAO,ER1)DIAOJIEmiRNAHUOXING-ceRNAZUOYONGMOSHIDEKEXINGXINGJIYANGXINGLVJIAOGAO,SHIMUQIANYANJIUZUIREMENDEcircRNAZUOYONGFANGSHI。

2、汉恒生物circRNA研究团队,为您提供基于ceRNA调控机制的circRNA系统研究方案。大致流程如下

1)YANZHENGcircRNADEBIAODASHUIPING;

2)TONGGUOWANGLUOSHUJUKU,YUCEcircRNAKENENGJIEHEDEmiRNAs;

3)circRNAJIEHEDEmiRNAsDEXINXIXUESHAIXUANJIYINGGUANGSUMEIJINYIBUSHAIXUANYANZHENG;

4)miRNABAJIYINYUCEYUGONGNENGFUJIFENXI;

5)miRNABAJIYINDESHAIXUAN、YANZHENGJIXIANGGUANGONGNENGYANJIU;

6)circRNA-miRNA-mRNADIAOKONGWANGLUODEYANZHENG。

 

TU2 HANHENGSHENGWUcircRNAYANJIULIUCHENGSHIYITU

 

三、汉恒生物-circRNA研究实例

circRNAMINGCHENG

hsa_circ_000244

YANJIUFANGXIANG

QIANLIEXIANAI

研究步骤解析:

1、验证circRNA的表达水平:

YIQIANLIEXIANAIZUZHIHEAIPANGZUZHIDEcDNAWEIYANGBEN,qPCRYANZHENGhsa_circ_000244DEBIAODALIANGJISHIFOUCHENGHUAN。

HANHENGSHENGWUYONGYOUZUICHENGSHUDEcircRNA-qPCRYANZHENGJISHU,KESHEJITESHUDEcircRNAYANZHENGYINWU,JIKEYANZHENGcircRNABIAODASHUIPING,TONGSHIYOUNENGQUFENCHENGHUANYUWEICHENGHUANRNA。

2、通过网络数据库,预测hsa_circ_000244可能结合的miRNAs:

 

 

 

3、circRNA结合的miRNAs的信息学筛选及荧光素酶进一步筛选验证:

JIANGhsa_circ_000244XULIEKELONGGOUJIANZHIBAOGAOJIYINZAITIpsiCHECK,HECHENGSHANGSHUYUCEDAODEmiRNA mimicsJIDUIZHAO,ZAI293TXIBAONEITONGSHIZHUANRANmiRNA mimicsHEpsiCHECK- hsa_circ_000244,DUOGONGNENGMEIBIAOYIJIANCEBAOGAOJIYINDEBIAODASHUIPING,BINGSHAIXUANBAOGAOJIYINBIAODAXIADIAODEmiRNAs(KENENGYOUDUOGE,BENLIZHONGJIADINGXUANZEhsa-miR-301a-3pJINXINGHOUXUYANJIU)。

4、miRNA靶基因预测与功能富集分析:

   对hsa-miR-301a-3p进行靶基因预测,结果如下:

 

 

通过功能富集分析将前列腺癌相关的基因筛选出来:

 

5、miRNA靶基因的筛选、验证及相关功能研究:

(1)CHUSHAI:qPCRYANZHENGSHANGSHUBAJIYINZAIQIANLIEXIANAIZUZHIHEAIPANGZUZHIZHONGDEBIAODALIANGCHAYI,SHAIXUANYUhsa_circ_000244BIAODACHENGZHENGXIANGGUANDEBAJIYIN(BENLIZHONGhsa_circ_000244ZAIAIZUZHIZHONGBIAODAJIAODI,YINCIXUYAOSHAIXUANZAIAIZUZHIZHONGBIAODALIANGJIAODIDEBAJIYIN);

(2)进一步筛选:在前列腺癌细胞系中过表达hsa_circ_000244(使用汉恒生物的超强成环系统HB circ-EnhancedTM circRNA慢病毒或腺病毒工具),qPCR验证上述靶基因表达水平。

汉恒生物自主研发的超强成环系统——HB circ-EnhancedTM circRNA环化载体系统(图谱如下),可获得全长过表达circRNA的腺病毒,慢病毒或腺相关病毒,适用于各种细胞和动物模型。

 

 

    XUANZEhsa_circ_000244GUOBIAODAHOU,BIAODALIANGYEMINGXIANSHANGDIAODEBAJIYINZUOWEIYANJIUDUIXIANG,BENLIZHONGJIADINGWEIPTENJIYIN。CHAXUNPTENDEGONGNENG,FAXIANTAZHUYAOYUXIBAOQINXIHEQIANYIXIANGGUAN。

(3)ZAIQIANLIEXIANAIXIBAOXIZHONGFENGBIhsa-miR-301a-3p(SHIYONGHANHENGSHENGWUDEMANBINGDUHUOXIANBINGDUGONGJU),WBHEqPCRJIANCEPTENDEBIAODASHUIPING;

(4)ZAIQIANLIEXIANAIXIBAOXIZHONGFENGBIhsa-miR-301a-3p(SHIYONGHANHENGSHENGWUDEMANBINGDUHUOXIANBINGDUGONGJU),TONGGUOXIBAOQIANYIHEXIBAOQINXIDENGSHIYAN,QUEDINGhsa-miR-301a-3pDUIXIBAOGONGNENGDEYINGXIANG;

(5)SHUANGYINGGUANGSUMEIBAOGAOJIYINSHIYAN:GOUJIANPTEN 3’UTRBAOGAOJIYINZAITI,JIANGhsa-miR-301a-3p mimicsYUBAOGAOJIYINZAITIGONGZHUAN293TGONGJUXIBAO,DUOGONGNENGMEIBIAOYIJIANCEBAOGAOJIYINDEBIAODASHUIPING,CHANMINGhsa-miR-301a-3pZUOYONGYUPTENDEFENZIJIZHI;

 

ZHU:WEILESHIYANDEYANJINXING,KEJIARUPTEN 3’UTRQUhsa-miR-301a-3pYUCEJIEHEQUYUDETUBIANTIZUOWEIDUIZHAO,JINYIBUSHUOMINGWENTI。

 

(6)QIANLIEXIANAIXIBAOXIZHONGGUOBIAODAhsa_circ_000244,JIANCEhsa-miR-301a-3pHEPTENDEBIAODASHUIPINGJIXIBAOGONGNENGXUEYANJIU:

在前列腺癌细胞系中过表达hsa_circ_000244(使用汉恒生物的超强成环系统HB circ-EnhancedTM circRNA慢病毒或腺病毒工具),然后分别进行如下实验:

1)WBHEqPCRJIANCEPTENBIAODASHUIPING;

2)qPCRJIANCEhsa-miR-301a-3pBIAODASHUIPING;

3)XIBAOQIANYIHEXIBAOQINXIDENGSHIYAN,YANJIUhsa_circ_000244DUIXIBAOGONGNENGDEYINGXIANG;

6、circRNA-miRNA-mRNADIAOKONGWANGLUODEYANZHENG:

HANHENGSHENGWU-circRNA-miRNA-mRNADIAOKONGJIZHIYANJIUJIEJUEFANGAN:JIYUceRNAZUOYONGFANGSHIHESHUANGYINGGUANGSUMEIBAOGAOJIYINSHIYAN,ZHENGMINGcircRNAKErescueXIAYOUmiRNADUIQIBAJIYINDEYIZHIZUOYONG。

BENSHILIDEJUTILIUCHENGRUXIA:

SHIYONGBUZHOU5(5)ZHONGGOUJIANDEPTEN 3’UTRBAOGAOJIYINZAITIHEBUZHOU5(2)ZHONGGOUJIANDEhsa_circ_000244GUOBIAODAZAITI,JIANGLIANGZHEGONGZHUANZHI293TGONGJUXIBAOZHONG,DUOGONGNENGMEIBIAOYIJIANCEBAOGAOJIYINDEBIAODASHUIPING,KEJIANJIEZHENGMINGhsa_circ_000244TONGGUOHAIMIANXIFUhsa-miR-301a-3p,CONGERSHANGDIAOPTENBAJIYINDEBIAODA。

ZHU:WEILESHIYANDEYANJINXING,KEJIARUPTEN 3’UTRQUhsa-miR-301a-3pYUCEJIEHEQUYUDETUBIANTIZUOWEIDUIZHAO,JINYIBUSHUOMINGWENTI。

四、参考文献:

1、Circular RNAs: diversity of form and function. Lasda E, Parker R. RNA. 2014 Dec;20(12):1829-42.

2、Circular RNAs are a large class of animal RNAs with regulatory potency. Memczak S, Jens M, Elefsinioti A, Torti F, Krueger J, Rybak A, Maier L, Mackowiak SD, Gregersen LH, Munschauer M, Loewer A, Ziebold U, Landthaler M, Kocks C, le Noble F, Rajewsky N. Nature. 2013 Mar 21;495(7441):333-8. 

3、Circular RNA profiling reveals an abundant circHIPK3 that regulates cell growth by sponging multiple miRNAs. Zheng Q, Bao C, Guo W, Li S, Chen J, Chen B, Luo Y, Lyu D, Li Y, Shi G, Liang L, Gu J, He X, Huang S.  2016 Apr 6;7:11215.

4、Circular RNA Related to the Chondrocyte ECM Regulates MMP13 Expression by Functioning as a MiR-136 'Sponge' in Human Cartilage Degradation. Liu Q, Zhang X, Hu X, Dai L, Fu X, Zhang J, Ao Y. Sci Rep. 2016 Mar 2;6:22572. D

 

 

汉恒生物-circRNA试用装申请

方式一:扫描下方二维码,申请汉恒生物-circRNA对照试用装。

                       

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