RNA实验技术

大约在十年前，2006年诺贝尔生理/医学奖得主CraigMello和AndrewFire发现，他们能够将短RNA 分子插入到线虫中并沉默特定基因的表达。今天，研究人员也常常使用这种强大的RNA 干扰方法来研究哺乳动物系统中的特定基因功能。 为了进行这种基因沉默实验，研究人员通常需要依赖化学合成的RNA 分子，而这个合成过程是相当昂贵的。本月《冷泉港实验手册》（ColdSpringHarborPro ...

本文来自于哈佛大学医学院果蝇RNAi 筛选中心的经典实验方法，专门用于果蝇RNAi 实验方法。感谢哈佛大学医学院果蝇RNAi 筛选中心的支持！ A.Primer Designed dsRNA B.Template Selection C.PCR D.In vitro RNA Transcription E.dsRNA Purif ...

N.B: Gloves should be worn at all times during preparation of in vitro RNA. All solutions should be RNase-free i.e. made with DEPC water if home-made or bought specifically to use for RNA work. You w ...

利用RACE ，可以很简单地得到mRNA的5＇末端序列，然而5＇RACE是一个很复杂的技术，它的成功依赖每一步反应的有效完成（图1）。cDNA第一链的合成是由一个反义的基因特异性引物（Gene－specific primer，GSP）来起始的，cDNA第一链纯化后，利用末端脱氧核苷酸转移酶（Terminal deoxynucleotidyl transferase，TdT）在cDNA的 ...

This is the preferred method for yeast RNA preparation use Gloves and RNAse free solutions throughout. 1. Use a YPD overnight culture to innoculate fresh YPD media and grow cells at 30 degrees overni ...

RNAi 实验中双链短RNA（dsRNA）制备过程，本实验方法来自于加州大学Jim教授实验，很权威！ Procedure for the Generation of dsRNA for use in RNAi 1.Design polymerase chain reaction (PCR ) primers that will amplify 600-800 bp of sequenc ...

3' R apid A mplification of c DNA E nds (RACE ) PCR This technique is used to obtain the 3'end of a cDNA it requires some sequence information internal to the mRNA under study. The sequence inf ...

T℃OS-M6 cells grow in 6-well plates 2ml media total T℃V1PD cells grow in 100mm plates. 1: Aspirate off the media from the cells and was the cell monolayers with 2x5ml (To 100mm dish) or 2x1ml (to 6-w ...

Add 0.1 ml DEPC to 100 ml of the solution to be treated and shake vigorously to bring the DEPC into solution. Let the solution incubate for 12 hours at 37℃. Autoclave for 15 minutes to remove any trac ...

Based on: Wan C.-Y. and Wilkins T.A. 1994. Anal. Bioch. 223:7-12. 1.Collect young expanding leaves (or other tissue) and freeze in liquid N2 and store at -80℃. 2.Pulverize tissue to a fine pow ...

1) Place gene of interest between T7 promoters in Òdouble T7Ó plamsid. (slightly more difficμlt alternative: Place gene of interest in hairpin/inverted repeat config ...

Gregory Hannon Cold spring harbor lab Our overall approach is to use an RNA polymerase III promoter to drive expression of encoded short hairpin RNA (shRNA). For this purpose we use the human U6 snRNA ...

pSico Oligo Design Click on the Mac OSX program pSicoOligomaker 1.5 to select and design oligos for pSico and plentilox3.7. To operate it simply paste your sequence in the "sequence" window ...

Preparation of Cells 1.Prepare or collect between 2x107 cells for each mRNA prep (will yield about 10-20µg of mRNA). If PBMCs from a whole blood sample are to be used the sample should be prepar ...

RNA干扰（RNA interference RNAi）正在迅速成为最受欢迎的阻断特异基因表达的技术。RNAi技术允许科学家关闭他们想要研究的基因，这是一种全新的令人激动的研究方法，通过研究基因表达缺失时细胞的表型，以确定基因的功能。最初，通过在无脊椎动物如：果蝇（Drosophila）和线虫（C. elegans）导入长的双链RNA，揭示了RNAi是一种降低特异基因表达的强有 ...

RNA Isolation - Volumes and weights are for 10 ml cultures (1-2 x 107 cells/ml). 1.Spin down cells decant and resuspend in 0.2 ml extraction buffer with SDS and transfer to a 1.5 ml eppendorf tu ...

Ambion and Applied Biosystems have joined forces to provide a complete convenient solution for performing gene silencing experiments and validating the results by real-time RT-PCR. Ambion's Silencer&t ...

1 ml linearized DNA template pBS-SK- ERD (X-E) (0.1-0.5 mg) 7.8 ml DDW 5 ml 5X transcription buffer ( stratagene ) 1 ml 750 mM DTT 1 ml Rnasin (RNase inhibitor; 15U/ml) 1 ml 10 mM ATP (pH 7) 1 ml 10 m ...

Steve Hahn. last modified Sat Oct 17 1998 Mix the following in an 0.5 ml eppendorf tube: 10-20 micrograms total yeast RNA 10 microliters hybridization mix H2O to bring the final volume to 25 microlite ...

1. 对每个基因设计并检测两到四个siRNA 序列 为了找到潜在靶位点，扫描靶基因中的AA序列。记录每个AA 3’端19个核苷酸作为潜在siRNA 靶位点。潜在靶位点需通过GENBANK数据库的BLAST分析，去除那些与其它基因明显同源的靶位点。如果可能，siRNA 应根据mRNA 低二级结构的区域设计。美国著名RNA 产品公司Ambion提供网上在线设计工具，帮助您更快地完成这项工作 ...