Northern 验证miR的方法目前看到的有三种标记探针：同位素、DIG和ECL。最多的是同位素、有文报道LNA－DIG与同位素效果差不多，ECL的结果好像不怎么理想。
不知道国内用DIG作标记的实验结果做得怎么样？
另外，由于没有条件做同位素，请教国内哪家公司代做或者提供做Northern场所？
战友：你好！我以前做过siRNA，对microRNA也很感兴趣，能将电子书发到我邮箱里吗？（cherry66@21cn.com)
谢谢！
MiRNA的检测方法：
1、Northern blot：此类文献已很多。
2、Solution hybridization （液相杂交）：经研究表明：液相杂交和Northern blot检测miR28均显出阳性结果，但液相杂交所用的细胞总RNA量(5 ug)明显少于Northern blot(30ug )，液相杂交的条带信号也较Northern blot的强。液相杂交是一种较Nothern blot更快速、简洁、敏感检测miR－28的方法。
徐卫，李建勇，陆凤翔。液相杂交检测B细胞淋巴瘤细胞系miR-28的表达。中国实验血液学杂志Journal ofExperimental Hematology 2006；14(2)：289-29
3、一个改良的Invader assay：用来定量分析miRNA,但是，这种方法缺乏特异性和灵敏性，而且，每次实验要求至少50 ng的总的RNA或者1000个裂解的细胞。
llawi, H.T., Dahlberg, J.E., Olson, S., Lund, E., Olson, M., Ma, W.-P., Takova, T., Neri, B.P., Lyamichev, V.I. (2004) Quantitation of microRNAs using a modified Invader assay RNA, 10, 1153–1161[Abstract/Free Full Text] .
4、Microarrays：能够高通量分析miRNA，但是，很难创建一个适合所有miRNA的杂交条件。
a、 一些Microarrays采用了用和premiRNA互补（而不是和成熟miRNA互补）的探针。然而，miRNA的成熟经常被调节，所以，premiRNA的水平并不总是和miRNA相一致。
b、 近年来，发展了的Microarrays采用和成熟miRNA序列特异结合的反义寡核苷酸作为探针来检测成熟miRNA，然而，相关miRNA杂交的问题仍未解决。另外，在反转录PCR、酶标或荧光标记衔接过程中还产生了系统偏差。
c、 以上问题能够通过RNA-primed array-based Klenow enzyme (RAKE) assay [92]. DNA寡核苷酸探针包含了和miRNA反义序列及通用的间隔序列。当miRNA和探针结合时，在加入了Klenow enzyme 后miRNA作为引物来延伸，并且产生了和掺入的标记的核苷酸构成的双链片段，而容易被检测。这个方法在两个相关的miRNA需要被分别分析的时候尤其适用，因为3‘段错配的miRNA不能够延伸。
需要指出的是，在大多数情况下，Microarrays不能够象northern blotting一样定量分析，因而，很难精确决定miRNA的相对丰度。
[92] Nelson, P.T. et al. (2004) Microarray-based, high-throughput gene expression profiling of microRNAs. Nat. Methods 1, 155–161
4、RT-PCR：
毫无疑问是检测miRNA最灵敏的方法，但是，如果miRNA的数目超过300就很难进行高通量的分析了。目前的方法是根据检测pre－miRNA而不是成熟的miRNA.[83]
[83] Schmittgen, T.D. et al. (2004) A high-throughput method to monitor.the expression of microRNA precursors. Nucleic Acids Res. 32, e43
5、the bead-based flow cytometric method：
最近的检测miRNA的方法就是[90].这个方法能够更加特异的检测相关连的miRNA。然而，此程序比较复杂。重要的是，在用此方法分析的218来自人类正常和肿瘤组织样本中217个已知的miRNA，阐明了miRNA和肿瘤的发育和分化有着高度准确的相关性。
【90】 Lu, J. et al. (2005) MicroRNA expression profiles classify human cancers. Nature 435, 834–838
对MiRNA干扰的方法：
1、通过引入和pri-miRNA, the pre-miRNA or the mature miRNA互补的反义miRNA寡核苷酸来抑制miRNA的活动
J Weiler1, J Hunziker1 and J Hall1 Anti-miRNA oligonucleotides (AMOs): ammunition to target miRNAs implicated in human disease? Gene Therapy (2006) 13, 496–502. doi:10.1038/sj.gt.3302654; published online 29 September 2005
2、通过RNAi来减少exportin5，进而减少了pre－miRNA，使得成熟miRNA的量减少。
Rui Yi2, Yi Qin3, Ian G. Macara3 and Bryan R. Cullen1,2,4 Exportin-5 mediates the nuclear export of pre-microRNAs and short hairpin RNAs. GENES & DEVELOPMENT 17:3011-3016, 2003
3、通过α-amanitin抑制了RNA聚合酶二的活性，进而减少了miRNA基因向pri-miRNAs 的转录，即减少了pri-miRNAs的生成。
MicroRNA genes are transcribed by RNA polymerase II. Yoontae Lee,1 Minju Kim,1 Jinju Han,1 Kyu-Hyun Yeom,1 Sanghyuk Lee,2 Sung Hee Baek,1 and V Narry Kim1a. EMBO J. 2004 October 13; 23(20): 4051–4060.
以上是我刚刚接触了的关于MiRNA的知识，其中涉及到的每种方法只挑选了一篇文献作为代表，因为自己也是读的文献有限，所以暂时自己就了解的这么多。衷心希望各位做MIRNA的战友多多发帖，咱们共同讨论，共同进步，共同提高！！
求助：最近读了一部分antisense inhibitor 来抑制miRNA表达的文章，其中有一段外文看不懂，各位高手能否给翻译一下并解释其内容，大家也可一同探讨。谢谢。文章如下：
(A) MiRNA reporter vectors used to analyze miRNA activity. The restriction sites for cloning the miRNA-binding sites are located in the 30-UTR of the luciferase gene in the pmir-Report luciferase vector. Once cloned the miRNA-binding site vector is co-transfected with the pmir-Report b-gal vector as a control for transfection efficiency and a miRNA inhibitor molecule that is specific to the binding site or is mutated as compared with the binding site. ( Enhanced expression of miRNA-regulated reporter by miRNA inhibitors. HeLa cells were plated at 50 000 cells/well in 24-well plates. Cells were transfected using lipofectamine 2000 in duplicate with pmir-REPORT b-gal, a luciferase reporter construct that contained one target site for either miR-23, miR-21, miR-15a, miR-16 or miR-19a and either inhibitors for these miRNA or a negative control (NC). HeLa cells were also transfected with a control luciferase reporter vector lacking a miRNA-binding site without an inhibitor to demonstrate the level of activity that can be achieved for an unmodified pmir-REPORT luciferase. Twenty-four hours post-transfection cells were assayed for luciferase and b-gal expression, and b-gal is used to normalize for differences in transfection efficiency.
microRNA高效克隆的新方法：mRAP
发表在：Nucleic Acids Res. 2006 Sep 14;
Mouse microRNA profiles determined with a new and sensitive cloning method.
Takada S, Berezikov E, Yama***a Y, Lagos-Quintana M, Kloosterman WP, Enomoto M, Hatanaka H, Fujiwara SI, Watanabe H, Soda M, Choi YL, Plasterk RH, Cuppen E, Mano H.
Division of Functional Genomics, Jichi Medical University 3311-1 Yakushiji, Shimotsukeshi, Tochigi 329-0498, Japan.
MicroRNAs (miRNAs) are noncoding RNA molecules of 21 to 24 nt that regulate the expression of target genes in a post-transcriptional manner. Although evidence indicates that miRNAs play essential roles in embryogenesis, cell differentiation and pathogenesis of human diseases, extensive miRNA profiling in cells or tissues has been hampered by the lack of sensitive cloning methods. Here we describe a highly efficient profiling method, termed miRNA amplification profiling (mRAP), as well as its application both to mouse embryos at various developmental stages and to adult mouse organs. A total of 77 436 Small-RNA species was sequenced, with 11 776 of these sequences found to match previously described miRNAs. With the use of a newly developed computational prediction algorithm, we further identified 229 independent candidates for previously unknown miRNAs. The expression of some of these candidate miRNAs was confirmed by northern blot analysis and whole-mount in situ hybridization. Our data thus indicate that the total number of miRNAs in vertebrates is larger than previously appreciated and that the expression of these molecules is tightly controlled in a tissue- and developmental stage-specific manner.
Facile means for quantifying microRNA expression by real-time PCR
下载地址：
>
到此一游，觉得不错，经常来看，读后在发表自己的意见。
[求助]刚刚接触RNAi技术,不知从何开始,版主建议一下看一些哪方面的资料
高手如云!
学习学习.
humana 得 microRNA protocols 我也有.欢迎大家索取.
goldideas
我是一名新生，刚刚接触到microRNA ,很需要humana 的 microRNA protocols
这本书，不知道可以发给我吗？
我的邮箱是 xialx@ioz.ac.cn
谢谢了
goldideas
你好,我也打算从事这方面的研究,请求把 microRNA protocols 也发给我一份,多谢!!! 邮箱:hnwangcj@yahoo.com.cn
好久都没有人讨论了;
坛子大了，酒也就不香了。
