RNA试验基础

Nature Biotechnology 5年关于RNAi专题 :http://www.dxy.cn/bbs/post/view?bid=64&id=3130708&sty=1&tpg=1&age=01 Synthetic shRNAs as potent RNAi triggers2 Synthetic dsRNA Dicer substrates enhance RNAi potency and efficacy.◎3 Knockdown by RNAi-proceed with caution4 Rati ...

miRNA已被证实与多种癌症的发生发展有关。某些特殊miRNA缺陷会导致其靶mRNA失去控制。目前，寻找miRNA靶点主要通过软件预测，但目前流行的预测算法所得到的结果中存在大量假阳性，或者无法获得有用的靶mRNA，这些结果会给后续验证实验造成很多麻烦。6月10日发布的Cancer Research，报道了第二军医大学、浙江大学医学院及中国医学科学院研究人员共同研发的一种名为miRIP（miRNA in vivo prec ...

慢病毒包装简要程序：以六孔板中的1孔为例，每个样品需要1×106个293FT细胞。1、取1.5ml灭菌EP管，加入1.5μg包装混合质粒和0.5μg表达质粒以及250μl的无血清Opti MEM。轻柔混匀，室温孵育5min。2、取1.5ml灭菌EP管，取9μl 脂质体2000l溶于250μl无血清Opti-MEM I培养基中。轻柔混匀，室温孵育5min。3、将DNA溶液和脂质体溶液轻柔混匀。室温孵育20min4、用胰酶消化并记数2 ...

快速的操作，低温环境，少量取上清。主要是抽提之后吸取上清时要特别小心，只要不把蛋白层吸出来就不会有RNase污染。分子克隆第二版343页,上关于提取RNA所用的玻璃容器的处理方法,是用前180度干烤8小时或更长时间;另一种方法是0.1%的depc水浸泡(37度2小时),然后灭菌水淋洗数次，100度干烤15分钟,然后高压灭菌除去depc。我们实验室一直是用170度考4小时，且不准离人，不准过夜,可能是怕烤箱长时间高温, ...

快速的操作，低温环境，少量取上清主要是抽提之后吸取上清时要特别小心，只要不把蛋白层吸出来就不会有 RNase 污染。分子克隆第二版343页,上关于提取RNA所用的玻璃容器的处理方法,是用前180度干烤8小时或更长时间;另一种方法是0.1%的depc水浸泡(37度2小时),然后灭菌水淋洗数次,100度干烤15分钟,然后高压灭菌除去depc.我们实验室一直是用170度考4小时,且不准离人,不准过夜,可能是怕烤箱长时间高温,线路 ...

1.在设计RNAi实验时，可以先在以下网站进行目标序列的筛选： http://www.ambion.com/techlib/misc/siRNA_finder.html http://katahdin.cshl.org:9331/RNAi/ http://www.ic.sunysb.edu/Stu/shilin/rnai.html2.RNAi目标序列的选取原则： （1）siRNAs with lower G/C content (35-55%) are more active than t ...

Small RNA是一大类调控分子，几乎存在于所有的生物体中。Small RNA包括：miRNA、ncRNA、siRNA、snoRNA、piRNA、rasiRNA等等。Small RNA通过多种多样的作用途径，包括mRNA降解、翻译抑制、异染色质形成以及DNA去除，来调控生物体的生长发育和疾病发生。Small RNA转录组测序是鉴定和定量解析small RNA的新方法和有力工具。Roche GS FLXTitanium 、IlluminaHi ...

样品中RNA的纯度及整体质量对于后续的实验有重要的影响。以下是进行PCR array前RNA质控的推荐标准：定量：NanoDrop/Spectrophotometer（用于检测样本中的核酸浓度，蛋白与核酸的比例及是否存在污染，例如有机物/盐离子等污染）。请记录样品的浓度，230，260及280的吸光值。此方法无法评估RNA的完整性。● 浓度：最佳浓度是100-150ng/uL。如果低于此浓度，可以使用PreAMP system或 ...

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3'RACE PCR3' Rapid Amplification of cDNA Ends (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 information obtained from this technique can be utilised to obtain full length cDNA clones using the 5'RA ...

The Easiest Route to Guaranteed Silencing Increasing numbers of researchers are using small interfering RNAs (siRNAs) to reduce the expression of specific mammalian genes. Applications of siRNA induced RNA interference (RNAi) include gene function analysis, pathway elucidat ...

We routinely produce dsRNA by in vitro transcription of a PCR generated DNA template containing the T7 promoter sequence on both ends (I. Primer Designed dsRNA). It is also possible to produce dsRNA using PCR generated DNA templates containing either the T7 & SP6 or the T7 & T3 promoters on either end (II. ds ...

Polymerase III in vitro TranscriptionSteve Hahnlast modified 10/15/99For the following reactions, use appropriate shielding and dispose of radioactive waste properly!A 20 microliter transcription reaction contains:4.0 ul 5X Pol III transcription buffer0.2 ul 0.1 M DTT0.2 ul a ...

Hot phenol may also be used to remove DNA. 1. Add equal volume of TE-saturated phenol to RNA solution. Mix. 2. Heat to 70 ℃ for 5 minutes. 3. Centrifuge at top speed for 10 minutes at room temperature. 4. Transfer the aqueous phase to a fresh tube. 5. Proceed to extract with cold phenol:chloroform:isoamyl alcohol. 上一篇： ...

Prepare in a sterile tube: template RNA:total RNA 0.1-5µgor poly(A)+mRNA 10ng-0.5µg,or specific RNA 0.01pg-0.5µg primer:oligo(dT)18 0.5µgor random hexamer 0.2µg,or sequence-specific 15-20pmol, deionized water (nuclease free) up to 11µl. Incubate the mix at 70°C for 5 minutes and chill on i ...

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 will need: 100mM DTT (Gibco-BRL)Ribonuclease inhibitor (RNasin, Pharmacia)T7 or T3 RNA Polymerase (Gi ...

一、 Riboclone M-MLV(H- ) cDNA合成技术 　　Promega公司的RibocloneR M-MLV(H- ) cDNA合成系统采用M-MLV反转录酶的RNase H缺失突变株取代AMV反转录酶,使合成的cDNA更长。该系统的第一链合成使用M-MLV反转录酶,cDNA第二链合成采用置换合成法,采用RNaseH和DNA聚合酶Ⅰ进行置换合成,最后用T4 DNA聚合酶切去单链末端,方法简便易行。该系统试剂包括：　　20μg 特异性引物 　　200μl M-MLV第 ...

To COS-M6 cells grow in 6-well plates, 2ml media totalTo CV1PD 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-well plates) ice cold TD (4oC is OK). Be very gentle when washing the cells as transfected cells tend to come off the dish e ...

Chemicals needed:GlucoseL-(+)-Arabinose Sigma Cat # A3256L-RhamnoseSigma cat # R3875Cb (carbenicillin) Sigma cat # 1389DL-p-Chlorophenylalanine Sigma cat # C6506Media Recipes:YEG recombination plates:1 liter Milli Q water5 g yeast extract10 g NaCl (5 g for any Zeocin recombin ...

Required Reagents:ChloroformIsopropanol75% Ethanol (cold)Monophasic Isolation ReagentRNA Precipitation Solution1.2M NaCl0.8M Disodium CitrateDNase (RNase-free)Ultra-pure waterLiquid NitrogenSolutions250ml RNA Precipitation Solution17.53 ...