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class="breadcrumbs">TechNotes</a> > <a href="index.html" class="breadcrumbs"><strong>Volume 12:3</strong></a></span></td> </tr> <tr> <td valign="middle"><table width="678" border="0" cellpadding="0" cellspacing="0" > <tr> <td colspan="2" bgcolor="003366" class="pagename"><table width="678" border="0" cellspacing="0" cellpadding="8"> <tr> <td class="pagename"><font color="ffffff">TechNotes 12(3)  </font><span class="ahead"><b><a name="top"></a></b></span> <div align="right"></div> </td> </tr> </table></td> </tr> <tr> <td width="452" valign="top"><table width="452" border="0" cellspacing="0" cellpadding="8"> <tr> <td valign="top" class="LIGHTleft"> <p align="left"><SPAN class="figure-label">MicroRNA Discovery</SPAN><BR> <SPAN class="ahead">MicroRNA Cloning Overview</SPAN></p> <p class="summary">MicroRNAs (miRNAs) are an important class of small RNAs that regulate protein translation (see <em><a href="11.html">Getting Started with MicroRNA Investigation</a></em>). To date, 230 mammalian miRNAs have been identified and cataloged in the <A href="http://microrna.sanger.ac.uk/sequences/index.shtml">Sanger miRNA Registry</A>. Some scientists believe that the number of miRNAs could exceed 1000 [1]. While cloning and sequencing were used to identify most of the known mammalian miRNAs, increasing effort is being put into bioinformatic prediction of new miRNA sequences. A combination of computational biology tools and molecular cloning methods will likely be used for characterizing additional miRNAs. </p> <p class="summary">Isolating the mature miRNA fraction is the first critical step in cloning these small regulatory molecules. Ambion scientists have developed two sequential systems that are optimized for isolating and enriching RNA preparations that contain 17–24 nucleotide, mature miRNAs. Here we describe the process by which miRNAs can be cloned in an effort to identify new members of this small RNA family.</p> <p> <SPAN class="basetextBOLD">1. Isolate total RNA using a method that will capture small RNAs </SPAN></p> <BLOCKQUOTE> <P><SPAN class="basetext"><em>•<a href="../../../catalog/CatNum.php?1560">mir</a></em><a href="../../../catalog/CatNum.php?1560">Vana miRNA Isolation Kit</a> includes small RNAs in the total RNA preparation and can subsequently be used to enrich for RNA <200 nt</SPAN></P> </BLOCKQUOTE> <p class="basetext"> <SPAN class="basetextBOLD">2. Separate mature miRNAs from precursor molecules</SPAN></p> <BLOCKQUOTE> <P class="basetext">•<a href="../../../catalog/CatNum.php?13100">flashPAGE Fractionator System</a> will quickly and easily purify mature miRNA</P> </BLOCKQUOTE> <p class="basetext"> <SPAN class="basetextBOLD">3. Ligate 3' donor oligonucleotide to RNA with T4 RNA ligase</SPAN></p> <BLOCKQUOTE> <P class="basetext">•The donor oligonucleotide may contain deoxynucleotides or both ribonucleotides and deoxynucleotides</P> <P class="basetext"> •This donor oligonucleotide should contain a 3' end blocking group (e.g., amino group, adenylation) to prevent self ligation and ligation to the RNA 5' ends</P> <P class="basetext"> •The donor oligonucleotide should be 5' phosphorylated</P> <P class="basetext"> •The donor oligonucleotide should contain a restriction enzyme site that is compatible with your cloning vector</P> <P class="basetext"> •Example: 5'-pCTGTA<strong>GGCACC</strong>ATCAAx-3' [2] (p=phosphorylation, bold type=restriction site, x=blocking group)</P> </BLOCKQUOTE> <p class="basetextBOLD"> 4. Gel purify RNA</p> <BLOCKQUOTE> <P class="basetext"> •Remove excess 3' oligonucleotides, enzymes, and salts</P> </BLOCKQUOTE> <p class="basetextBOLD"> 5. Ligate 5' acceptor oligonucleotide to RNA with T4 RNA ligase</p> <BLOCKQUOTE> <P class="basetext"> •This acceptor oligonucleotide will contain both ribonucleotides and deoxynucleotides</P> <P class="basetext"> •The acceptor oligonucleotide should contain the same restriction enzyme site that is in the 3' donor oligonucleotide</P> <P class="basetext"> •Example: 5'-ATCGTa<strong>ggcacc</strong>tgaaa-3' [2] (upper case=DNA, lower case= RNA, bold type=restriction site)</P> </BLOCKQUOTE> <p class="basetextBOLD"> 6. Gel purify RNA</p> <BLOCKQUOTE> <P class="basetext"> •Remove excess 5' oligonucleotides, enzymes, and salts</P> </BLOCKQUOTE> <p class="basetextBOLD"> 7. Use RT-PCR to amplify RNA</p> <BLOCKQUOTE> <P class="basetext">• RT and PCR primers are based on acceptor and donor oligonucleotide sequences used above</P> </BLOCKQUOTE> <p class="basetextBOLD"> 8. Digest with restriction enzyme whose target site was incorpo-<br>   rated into donor and acceptor oligonucleotides</p> <p class="basetextBOLD"> 9. Concatamerize PCR products with T4 DNA ligase and fill in the<br>   ends of the PCR products</p> <p class="basetextBOLD">10. Isolate ligation products (400–600 nt range) by gel purification</p> <BLOCKQUOTE> <P class="basetext"> •Resulting clones contain 4–6 inserts and provide longer templates for efficient sequencing analysis</P> </BLOCKQUOTE> <p class="basetext"><SPAN class="basetextBOLD">11. Clone into your cloning vector of choice</SPAN></p> <p class="basetextBOLD">12. Screen for vectors containing inserts by PCR</p> <p class="basetextBOLD">13. Purify vector/insert and sequence inserts</p> <em><p> <SPAN class="copyright">Additional details have been described by leading miRNA researchers [2–5].</SPAN></em> <p class="bhead">Summary</p> <p class="basetext">This is one method that has been used to uncover new miRNAs. When moving into validation and functional studies, we recommend using <a href="../../../catalog/CatNum.php?17100">Pre-miR miRNA Precursor Molecules</a> and <a href="../../../catalog/CatNum.php?17000">Anti-miR miRNA Inhibitors</a> to alter miRNA activity in a cell system. See <A href="../../../miRNA">www.ambion.com/miRNA</A> for more information.</p> <p> <span class="patent"><a href="#top">back to top</a></span><br> <br> <a name="ordering" id="ordering"></a> </p> </td> </tr> </table>   <table width="100%" cellpadding="4" cellspacing="0" border="0"> <tr> <td class="LIGHTleft"><p><span class="chead"><strong>Ordering Information for Ambion Products:</strong></span> </td> </tr> </table> <table width="100%" cellpadding="4" cellspacing="0" border="0"> <tr> <td bgcolor="#cccccc" class="figure-title"><b>Cat#</b></td> <td bgcolor="#cccccc" class="figure-title"><b>Product Name</b></td> <td bgcolor="#cccccc" class="figure-title"><b>Size</b></td> </tr> <tr> <td bgcolor="#eeeeee" valign="top" class="figure-text"><a href="../../../catalog/CatNum.php?AM10010">AM10010</a></td> <td bgcolor="#eeeeee" valign="top" class="figure-text" class="figure-text">flashPAGE™ Pre-cast Gels (Type A)</td> <td bgcolor="#eeeeee" valign="top" class="figure-text" class="figure-text">10 gels</td> </tr> <tr> <td bgcolor="#eeeeee" valign="top" class="figure-text"><a href="../../../catalog/CatNum.php?AM12200">AM12200</a></td> <td bgcolor="#eeeeee" valign="top" class="figure-text" class="figure-text">flashPAGE™ Reaction Clean-Up Kit</td> <td bgcolor="#eeeeee" valign="top" class="figure-text" class="figure-text">20 rxns</td> </tr> <tr> <td bgcolor="#eeeeee" valign="top" class="figure-text"><a href="../../../catalog/CatNum.php?AM13100">AM13100</a></td> <td bgcolor="#eeeeee" valign="top" class="figure-text" class="figure-text">flashPAGE™ Fractionator Apparatus</td> <td bgcolor="#eeeeee" valign="top" class="figure-text" class="figure-text">1 unit</td> </tr> <tr> <td bgcolor="#eeeeee" valign="top" class="figure-text"><a href="../../../catalog/CatNum.php?AM1560">AM1560</a></td> <td bgcolor="#eeeeee" valign="top" class="figure-text" class="figure-text"><i>mir</i>Vana™ miRNA Isolation Kit</td> <td bgcolor="#eeeeee" valign="top" class="figure-text" class="figure-text">up to 40 purifications</td> </tr> <tr> <td bgcolor="#eeeeee" valign="top" class="figure-text"><a href="../../../catalog/CatNum.php?AM9015">AM9015</a></td> <td bgcolor="#eeeeee" valign="top" class="figure-text" class="figure-text">flashPAGE™ Buffer Kit (Type A)</td> <td bgcolor="#eeeeee" valign="top" class="figure-text" class="figure-text">20 rxns</td> </tr> <tr> <td class="LIGHTleft" colspan="3"><span class="summary">For Research Use Only. Not for use in diagnostic procedures.</span></td> </tr> </table> </td> <td width="226" valign="top" bgcolor="#EEEEEE" class="LIGHTleftRIGHT"><table width="95%" border="0" align="center" cellpadding="12" cellspacing="0"> <tr> <td class="LIGHTbottom"><a href="../../Documents.html?fkResSxn=9&fkSubSxn=6" class="chart-heads">TechNotes Archive</a></td> </tr> <tr> <td class="LIGHTbottom"><a href="#ordering" class="figure-text">Ordering Information</a> </td> </tr> <tr> <td class="LIGHTbottom"> <p class="sidebarHEAD">References</p> <p class="sidebarBODY">1. Berezikov E, Guryev V, van de Belt J, Wienholds E, Plasterk RHA, Cuppen E. (2005) Phylogenetic shadowing and computational identification of human microRNA genes. <em>Cell</em><strong> 120(1):</strong>21–4.</p> <p class="sidebarBODY">2. Lau NC, Lim LP, Weinstein EG, Bartel DP. (2001) An abundant class of tiny RNAs with probable regulatory roles in <em>Caenorhabditis elegans</em>. <em>Science</em><strong> 294(5543):</strong>858–62.</p> <p class="sidebarBODY">3. Elbashir SM, Lendeckel W, Tuschl T. (2001) RNA interference is mediated by 21- and 22-nucleotide RNAs. <em>Genes Dev</em><strong> 15(2):</strong>188–200.</p> <p class="sidebarBODY">4. Lagos-Quintana M, Rauhut R, Lendeckel W, Tuschl T. (2001) Identification of novel genes coding for small expressed RNAs. <em>Science</em><strong> 294(5543):</strong>853–858.</p> <p class="sidebarBODY">5. Lee RC, Ambros V. (2001) An extensive class of small RNAs in <em>Caenorhabditis elegans</em>. <em>Science</em><strong> 294(5543):</strong>862–864</p> </td> </tr> <tr> <TD> <p align="left"><span class="sidebarHEAD">Related Links:</span><br> <table width="100%" cellpadding="0" cellspacing="0" border="0"> <tr> <td class="figure-text">miRNA Resource <a href="../../../techlib/resources/miRNA/index.html"><br>[read]</a><br><br></td> </tr> <tr> <td class="figure-text">Ultra-Fast Purification of Small Nucleic Acids <a href="../../../techlib/tn/122/2.html"><br>[read]</a><br><br></td> </tr> <tr> <td class="figure-text">Getting Started with microRNA Research <a href="../../../techlib/tn/123/5.html"><br>[read]</a><br><br></td> </tr> <tr> <td class="figure-text">Examine microRNA Profiles from Archived Formalin-fixed, Paraffin-embedded (FFPE) Tissue <a href="../../../techlib/tn/123/7.html"><br>[read]</a><br><br></td> </tr> </table> </td> </tr> </table> </td> </tr> </table></td> </tr> <TR> <TD bgcolor="#003366"> </TD> </TR> <TR> <TD>  <table width="100%" border="0" cellpadding="10" cellspacing="0"> <tr> <td class="LIGHTbottom" > <a class="copyrightBOTTOM" href="/main/search.html"> </a> <TABLE width="100%" border="0" cellspacing="0" cellpadding="1"> <TR> <TD class="copyrightBOTTOM"><A class="copyrightBOTTOM" href="/">Home</A> | <A class="copyrightBOTTOM" href="/catalog/">Products</A> | <A class="copyrightBOTTOM" href="/techlib/">Technical Resources</A> | <A class="copyrightBOTTOM" href="/news/">What's New</A> | <A class="copyrightBOTTOM" href="/about/">About Us</A> | <A class="copyrightBOTTOM" href="/contact">Contact Us</A> <A class="copyrightBOTTOM" href="/main/search.html"> </A></TD> </TR> <TR> <TD class="copyrightBOTTOM"><A class="copyrightBOTTOM" href="/main/search.html">Advanced Search</A> | <A class="copyrightBOTTOM" href="/main/sitemap.html"> Site Map</A> | <A class="copyrightBOTTOM" href="/about/direction.html#privacy">Privacy</A> | <A class="copyrightBOTTOM" href="/main/trademarks.html">Trademarks/Legal</A> | <A class="copyrightBOTTOM" href="/contact/web_feedback.html">Web Feedback</A> | <A class="copyrightBOTTOM" href="/about/jobs/index.html">Jobs</A> </TD> </TR> </TABLE> </td> </tr> <tr bgcolor="#FFFFFF"> <td class="copyrightBOTTOM" ><FONT color="#A9A9A9"><I>©<SCRIPT language="Javascript">  </SCRIPT> Applied Biosystems. 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