1.miRNA琛ㄨ揪鎴栬€呬笂璋冪殑鐮旂┒

a.miRNA杩囪〃杈捐浇浣?/font>
鐢≒CR鏂规硶灏唌iRNA鍩哄洜缁勫簭鍒楀厠闅嗗埌杞戒綋涓紝鐒跺悗灏嗙粨鍚堜綅鐐?'UTR鍖哄厠闅嗗埌Luciferase Reporter Vectors杩涜琛ㄨ揪鐮旂┒

b.鐢╩icroRNA mimics鏈変袱绉嶅舰寮?/strong>
I. microRNA鍙岄摼妯℃嫙鐗╋細鍙互妯℃嫙microRNA鐨勪笂璋冧綔鐢ㄣ€?br />II.microRNA precursor妯℃嫙鐗╋紝涓€鑸琣mbion鍏徃鏈夎繖鏍风殑鍓嶄綋妯℃嫙鐗╁悎鎴?/p>

2.鎶戝埗
鎶戝埗涔熸湁涓ょ褰㈠紡
I.閽堝鎴愮啛浣撶殑鎶戝埗
鐢╩icroRNA mature鐨勭殑浜掕ˉ閾捐繘琛屽叏鐢叉哀鎴栬€呭叾浠栫殑淇グ杩涜鎶戝埗microRNA鐨勪綔鐢?br />琛ュ厖璇存槑锛?br />Targeting of miRNAs with antisense oligonucleotides
Antisense oligonucleotide (ASO) inhibition is a powerful technique for studying miRNA function in vitro as well as in vivo, and for therapeutic targeting of miRNAs. Identification of optimal ASO chemistries for targeting miRNAs is important for this purpose. A number of 2'-sugar and backbone ASO modifications were evaluated for their ability to inhibit miR-21 activity on a luciferase reporter mRNA (Davis et al 2006). ASO modifications that improved target affinity improved miRNA ASO activity, but the positioning of highaffinity modifications also had dramatically different effects on miRNA activity, suggesting that factors other than affinity determined the effectiveness of the miRNA ASOs. To be an effective miRNA ASO, it may also be necessary to avoid dissociation by the factors responsible for si/miRNA passenger strand unwinding. The activity of a modified miRNA ASO was inversely correlated to its tolerability as a siRNA passenger strand, suggesting that a similar mechanism could be involved in the dissociation of miRNA ASOs and siRNA passenger strands. These studies begin to define the factors important for designing improved miRNA ASOs, enabling more effective miRNA functionalization and therapeutic targeting.

Silencing miRNAs by antagomirs
Scientists at Alnylam and the Rockefeller University have shown that a novel class of chemically engineered oligonucleotides, termed 'antagomirs', are efficient and specific silencers of endogenous miRNAs in mice. These antagomirs have remarkable properties including high potency, high specificity, broad bioavailability, and long-lasting effects. Intravenous administration of antagomirs against miR-16, miR-122, miR-192 and miR-194 results in a marked reduction of corresponding miRNA levels in liver, lung, kidney, heart, intestine, fat, skin, bone marrow, muscle, ovaries and adrenals (Krützfeldt et al 2005). The biological significance of silencing miRNAs with the use of antagomirs was studied for miR-122, an abundant liver-specific miRNA. Gene expression and bioinformatic analysis of mRNA from antagomir-treated animals revealed that the 3' untranslated regions of upregulated genes are strongly enriched in miR-122 recognition motifs, whereas downregulated genes are depleted in these motifs. Analysis of the functional annotation of downregulated genes specifically predicted that cholesterol biosynthesis genes would be affected by miR-122, and plasma cholesterol measurements showed reduced levels in antagomir-122-treated mice. These findings show that antagomirs are powerful tools to silence specific miRNAs in vivo and may represent a therapeutic strategy for silencing miRNAs in diseases such as cancer and viral infections

II閽堝鍓嶄綋鐨勬姂鍒?/strong>
鍙互鐢╯iRNA鎴栬€卻hRNA鎴栬€卨entivirus RNAi閽堝鍓嶄綋鑼庣幆缁撴瀯璁捐杩涜RNAi浣滅敤鎶戝埗microRNA

3.Morpholinos鍙嶄箟RNA鏂规硶锛?/strong>
鐜板湪涔熸湁鐢╣enetool鐨勫弽涔夋牳閰告妧鏈紝鐗规畩鐨勫崟浣撳悎鎴愭牳閰歌繘琛屾姂鍒躲€?br />Knockdowns of miRNA activity have generally used oligos targeting the miRNA guide strand. A Morpholino oligo targeting an miRNA guide strand can interfere with the activity of the miRNA. It is difficult to control for the specificity of the knockdown when using this technique alone. However, Morpholinos targeting the nucleolytic processing sites of an immature miRNA can prevent maturation of the miRNA. This allows sets of nonoverlapping Morpholino oligos targeting a primary miRNA to be used as specificity controls; if two non-overlapping oligos targeting the same miRNA produce the same phenotype, this supports the hypothesis that the phenotype is due to knocking down the activity of the targeted miRNA and not due to an off-target effect. These techniques are explored in the following paper:
Kloosterman WP, Lagendijk AK, Ketting RF, Moulton JD, Plasterk RHA. Targeted inhibition of miRNA maturation with morpholinos reveals a role for miR-375 in pancreatic islet development. PLoS Biol. 2007;5(8): e203.
Available online:

Nature paper:
MicroRNA control of Nodal signalling.
鐜板湪鍙戦珮妗ｆ鏂囩珷涓€鑸槸杩欎簺鎶€鏈殑缁煎悎缁撴灉鎵嶈兘璇存槑闂锛?/p>

浜屻€乵icroRNA妫€娴嬬殑鏂规硶锛?/strong>
microRNA妫€娴嬩粠妫€娴嬬殑鏂规硶瀛︽垜褰掔撼涓婃湁涓ょ锛?br />1.鎺㈤拡娉?br />2.鏌撴枡娉?/font>

1.鎺㈤拡娉曪細
ABI鎻愪緵microRNA assay Kit:
a.閫嗚浆褰曞紩鐗㏑T-Primer鐗瑰紓鎬?/strong>
鐗瑰紓鎬т富瑕佸湪涓や釜鏂归潰锛歭oop鐜殑鍙嶈浆褰曞紩鐗╂彁楂樼壒寮傛€с€俆aqman鐨凪GB鎺㈤拡鎻愰珮妫€娴嬬殑鐏垫晱搴﹀拰鐗瑰紓鎬с€備环鏍间富瑕佸湪鎺㈤拡鍚堟垚涓娿€侫BI浜у搧鐜板湪鏈夌浉瀵逛究瀹滅殑灏忓寘瑁咃紝鐜板湪杩欑鏂规硶宸茬粡鐢ㄤ簬鍗曠粏鑳濸CR鎴栬€呭師浣峆CR绛夌瓑

b.鐗瑰紓鐨刴icroRNA primer sets and probe

c.LNA probe detection kit

2.鏌撴枡娉?/font>
a.涓昏鐢ㄧ壒寮傜殑microRNA primer sets缁撳悎SYBR鏌撴枡妫€娴?/strong>
ABI鍜孖nvitrogen閮芥湁鐩稿簲鐨勮瘯鍓傜洅
鏂规硶鐗瑰紓鎬у湪浜庡紩鐗╂帴澶磋璁★紝鎴戜釜浜鸿涓鸿繖绉嶆柟娉曟垚鏈綆锛屾湁浜涙晥鏋滆繕鏄笉閿欙紝瀵逛簬涓€鑸殑鐢ㄦ埛杩樻槸鍙互鐨勩€?/p>

b鍔犱簡LNA鐨勫崟浣撶殑寮曠墿锛屾彁楂樼殑TM鍊笺€備絾鏄悎鎴愬拰璁捐LNA鐨勫紩鐗╄繕鏄渶瑕佽垂蹇冩€濈殑銆?/strong>

c.鏌撴枡娉曟娴媘icroRNA鏂规硶杩樺緢澶氾紝鍏蜂綋鐨勬柟娉曞彲浠ュ弬鑰冩枃鐚紝涓昏鍦ㄤ簬閫嗚浆褰曠殑鏂规硶浠ュ強鐗瑰紓鐨勫紩鐗╃殑璁捐绛夌瓑锛屽ぇ瀹舵湁濂界殑鏂囩尞浠ュ強鎬濊矾涔熷彲浠ョ晠鎵€娆茶█

microRNA妫€娴嬩篃鍒嗗墠浣撳拰鎴愮啛浣撴娴嬶紝鍏蜂綋鏂规硶鍙互鍙傝€冨悇鑷殑妫€娴嬪師鐞嗘潵璋冩暣銆?/p>

3.Microarrays for analysis of miRNA gene expression

 microRNA鐨勬柟娉曟潵璇寸幇鍦ㄤ竴鑸敤LNA鎺㈤拡鏇村叿鍑嗙‘鎬?/p>

4.缁忓吀鐨刵orthern blot鏂规硶杩涜妫€娴?/strong>

鐜板湪寰堝璇曢獙鐢ㄥ悓浣嶇礌杩涜northern blot妫€娴媘icroRNA銆?/p>

涓夈€乵iroRNA棰勬祴浠ュ強闈剁偣鍒嗘瀽

Computational approaches are important for identification of miRNAs as well as
prediction of their gene targets. Various computational tools and databases have been
reviewed recently (Brown and Sanseau 2005). There is a searchable database called
miRNA Registry, which is maintained at the Sanger Centre in the UK
().

microRNA 鐮旂┒缁嗚妭杩囩▼鍙互鍙傝€僲icroRNA protocol book

杩欓噷鐨勬€荤粨鏈夐檺锛屽笇鏈涘ぇ瀹舵祻瑙堟垜鍗氬鐨勫悓浠佸澶氫氦娴佷互鍙婅ˉ鍏呫€?/p>