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实验目的 从黄化小麦苗中提取总RNA ，可以为cDNA 文库的构建做好准备。我们重点是要保证RNA 的完整性、产率、防止修饰和纯度，尤其是完整性、防止修饰和纯度。RNA 是单链，2’OH是它的化学性质远比DNA 活泼。所以，提取RNA 颇为不易。加之RNA 的不均一性，要将所有的RNA （rRNA 、tRNA 、mRNA ）完全提出，更加有挑战性。而Rnase是无处不在的（这种酶遇高 ...

真核细胞的mRNA分子最显著的结构特征是具有5’端帽子结构（m7 G）和3’端的Poly(A)尾巴。绝大多数哺乳类动物细胞mRNA的3’端存在20-30个腺苷酸组成的Poly（A）尾，通常用Poly（A+ ）表示。这种结构为真核mRNA的提取，提供了极为方便的选择性标志，寡聚（dT）纤维素或寡聚（U）琼脂糖亲合层析分离纯化mRNA的理论基础就在于此。 mRNA的 ...

RNA 酶污染是全体从事 RNA 相关实验的人员的头号公敌。即使是100度高温或者是高压灭菌也不能完全灭活无孔不入的 RNases 。因而相关的实验中往往要用到RNA 酶抑制剂。最常用的 RNA 酶抑制剂 Human Placental ribonuclease inhibitor （ HPRI ）是一种蛋白质，可与多种 RNA 酶（包括 RNases A B C 类）非共价结合成为等摩尔复合物， ...

小分子干扰RNA (Small interfering RNA ，siRNA ) 是一种非常有效的工具，能够在包括哺乳动物细胞在内的多个体系中抑制特定基因的表达，从而研究某个基因的功能或者是相关信息。但是这种强有力的方法的难题之一是需要设计，合成siRNA s 和验证其效果，从而找到最有效的siRNA s 。这个过程需要花费相当的时间和经费。以化学法合成基因特异的siRNA s 为例，由于设计好的 ...

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 ...

Here is our T7 siRNA protocol. The main idea is to design primers that begin with GG so that transcription by T7 polymerase is efficient. I) FIRST design oligos: Using sense coding sequence of any gen ...

Helvetica sans-serif" size="-1"Our current strategy with siRNA is to synthesis relatively small amounts enzymatically and use these to test for efficiency by western blotting. This allows us to test ...

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 u ...

Prior to Extractions ・ Bake all necessary glassware metal spatulas mortars and pestles overnight in a 200°C oven after wrapping them in aluminum foil. For each sample you should minima ...

AMES/Chloroform extraction in our lab to extract total from potato leaves for viroid analysis. The protocol is as follows: AMES Buffer (for 200mL): 11.7g NaCl 160 mL dH2O &nbs ...

Restriction Map and Cloning Site of the RNAi-Ready pSIREN-DNR Vector. Unique restriction sites are in bold. RNAi-Ready pSIREN-DNR is pr ...

3' 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 obt ...

Post-transcriptional gene silencing (PTGS) which was initially considered a bizarre phenomenon limited to petunias and a few other plant species is now one of the hottest topics in molecular biology ( ...

Supplementary information to miRNAs cloned from mouse and human An online clearinghouse for miRNA gene name assignments (http://www.sanger.ac.uk/Software/Rfam/mirna/) is provided by the Rfam database ...

Note: Start with 20 mg of total RNA for each labeling reaction. All solutions that can be filtered should be filtered. Cy dyes are light sensitive and should ALWAYS be handled in dim light. RNA ...

Prepare in a sterile tube: template RNA: total RNA 0.1-5µg or poly(A)+ mRNA 10ng-0.5µg or specific RNA 0.01pg-0.5µg primer: oligo(dT)18 0.5µg or ...

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 induce ...

The following protocol is for MEGAscript II Kit (Ambion). 1. PCR amplify the DNA template. The 5'-end of the template should contain the minimum promoter sequenences of T7 or Sp6 or T3. A 5-primer wi ...

in vitro Transcription Reaction 1 µl 10X Transcription Buffer (Ambion) 1 µl 10X NTPs (4 mM ATP CTP 1 mM GTP UTP) 2 µl 10 mM GpppG cap (Pharmacia) 2 µl a-UTP (NEN) 800 Ci/mmo ...

The siRNA user guide (revised May 6 2004) Selection of siRNA duplexes from the target mRNA sequence Using Drosophila melanogaster lysates (Tuschl et al. 1999) we have systematically ...