Physiology & Endocrinology & Gene Quantification & real-time PCR

</head> <body text="#000000" bgcolor="#FFFFFF" link="#0000EE" vlink="#FF0000" alink="#FF0000" background="dna-back.gif"> <center><a NAME="top"></a></center> <table CELLSPACING=0 CELLPADDING=0 WIDTH="100%" > <tr ALIGN=LEFT VALIGN=TOP> <td ALIGN=CENTER VALIGN=TOP WIDTH="150" BGCOLOR="#000099"> <center> <br><a href="http://www.tu-muenchen.de/" target="_blank"><img SRC="logo-tum.gif" NOSAVE BORDER=0 height=123 width=98></a> <p><a href="http://www.wzw.tum.de/gene-quantification/main.shtml" target="main"><img SRC="button-home.gif" ALT="gene.quantification@wzw.tum.de" height=20 width=140></a><a href="http://www.wzw.tum.de/gene-quantification/mrna.html" target="main"><img SRC="button-mrna.gif" ALT="mRNA structure and analysis methods" BORDER=0 height=20 width=140></a><a href="http://www.wzw.tum.de/gene-quantification/block.html" target="main"><img SRC="button-block-pcr.gif" ALT="classical block RT-PCR" height=20 width=140></a><a href="http://www.wzw.tum.de/gene-quantification/competitive.html" target="main"><img SRC="button-comp.gif" ALT="competitive RT-PCR" height=20 width=140></a><a href="http://www.wzw.tum.de/gene-quantification/real-time.html" target="main"><img SRC="button-real-time.gif" ALT="real-time RT-PCR" height=20 width=140></a><a href="http://www.wzw.tum.de/gene-quantification/platform.html" target="main"><img SRC="button-platform.gif" ALT="available real-time PCR platforms" height=20 width=140></a><a href="http://www.wzw.tum.de/gene-quantification/optimization.html" target="main"><img SRC="button-optim.gif" ALT="optimization strategies for kinetic RT-PCR" BORDER=0 height=20 width=140></a><a href="http://www.wzw.tum.de/gene-quantification/strategy.html" target="main"><img SRC="button-strategy.gif" ALT="real-time RT-PCR quantification strategies" height=20 width=140></a><a href="http://www.wzw.tum.de/gene-quantification/absolute.html" target="main"><img SRC="button-absolute.gif" ALT="absolute quantification using real-time PCR" height=20 width=140></a><a href="http://www.wzw.tum.de/gene-quantification/relative.html" target="main"><img SRC="button-relative.gif" ALT="relative quantification using real-time PCR" height=20 width=140></a><a href="http://www.wzw.tum.de/gene-quantification/rest.html" target="main"><img SRC="button-rest.gif" ALT="Relative Expression Software Tool (C)" height=20 width=140></a><a href="http://www.wzw.tum.de/gene-quantification/download.html#rest" target="main"><img SRC="button-rest-xl.gif" ALT="Relative Expression Software Tool (C)" height=20 width=140></a><a href="http://www.wzw.tum.de/gene-quantification/download.html#rest" target="main"><img SRC="button-download.gif" ALT="download of software tools" height=20 width=140></a><a href="http://www.wzw.tum.de/gene-quantification/hkg.html" target="main"><img SRC="button-hkg.gif" ALT="problems derived from housekeeping genes" A LT="housekeeping genes" height=20 width=140></a><a href="http://www.wzw.tum.de/gene-quantification/array.html" target="main"><img SRC="button-array.gif" ALT="verification of array results by real-time RT-PCR" height=20 width=140></a><a href="http://www.wzw.tum.de/gene-quantification/endo.html" target="main"><img SRC="button-endo.gif" ALT="real-time PCR applications - Endocrinology" height=40 width=140></a><a href="http://www.wzw.tum.de/gene-quantification/endo.html#erab" target="main"><img SRC="button-receptors.gif" ALT="real-time PCR applications - Steroid Receptors" height=20 width=140></a><a href="http://www.wzw.tum.de/gene-quantification/endo.html#adrenerg" target="main"><img SRC="button-adrenergic.gif" ALT="real-time PCR applications - Adrenergic Receptors" height=20 width=140></a><a href="http://www.wzw.tum.de/gene-quantification/endo.html#igfs" target="main"><img SRC="button-somatotropic.gif" ALT="real-time PCR applications - Somatotripic Axis - IGFs and IGF-BPs" height=20 width=140></a><a href="http://www.wzw.tum.de/gene-quantification/endo.html#serotonin" target="main"><img SRC="button-serotonin.gif" ALT="real-time PCR applications - Serotonin Receptors" height=20 width=140></a><a href="http://www.wzw.tum.de/gene-quantification/immuno.html" target="main"><img SRC="button-immunology.gif" ALT="real-time PCR applications - Mammary Gland Immunology" height=40 width=140></a><a href="literature.html" target="main"><img SRC="button-literature.gif" ALT="real-time PCR literture" height=20 width=140></a><a href="poster-a.html" target="main"><img SRC="button-poster.gif" ALT="visit our kinetic PCR poster board" height=20 width=140></a><a href="http://www.wzw.tum.de/gene-quantification/link.html" target="main"><img SRC="button-link.gif" ALT="links to WWW" height=20 width=140></a><a href="http://www.wzw.tum.de/gene-quantification/link.html#news" target="main"><img SRC="button-news.gif" ALT="links to WWW" height=20 width=140></a><a href="http://www.wzw.tum.de/gene-quantification/movie.html" target="main"><img SRC="button-movie.gif" ALT="animations and movies" height=20 width=140></a><a href="http://www.wzw.tum.de/gene-quantification/contact.html" target="main"><img SRC="button-contact.gif" ALT="contact responsible author" height=20 width=140></a><a href="http://www.wzw.tum.de/gene-quantification/contact.html#server" target="main"><img SRC="button-server.gif" ALT="page and download frequency" height=20 width=140></a><a href="http://www.wzw.tum.de/gene-quantification/copyright.html" target="main"><img SRC="button-copyright.gif" ALT="(C) copyright of gene.quantification@wzw.tum.de and OTHERS" height=20 width=140></a><a href="http://www.wzw.tum.de/gene-quantification/main.shtml" target="main"><img SRC="button-home.gif" ALT="gene.quantification@wzw.tum.de" height=20 width=140></a> <br> <br><a href="http://www.tu-muenchen.de" target="_blank"><img SRC="button-tum.gif" ALT="Technical University of Munich" height=20 width=140></a><a href="http://www.weihenstephan.de/wzw/function.html" target="_blank"><img SRC="button-wzw.gif" ALT="Center of Life Sciences Weihenstephan" height=20 width=140></a><a href="http://www.edv.agrar.tu-muenchen.de/blm/fml/index.html" target="_blank"><img SRC="button-fml.gif" ALT="Research Center for Milk and Food" height=20 width=140></a><a href="http://www.edv.agrar.tu-muenchen.de/fml/physio/index.html" target="_blank"><img SRC="button-physiology.gif" ALT="Institute of Physiology" height=20 width=140></a><a href="http://www.wzw.tum.de/gene-quantification/main.shtml" target="main"><img SRC="button-home.gif" ALT="gene.quantification@wzw.tum.de" height=20 width=140></a></center> <p><br> <br> <br> <br> <p> </td> <td ALIGN=LEFT VALIGN=TOP> <center><a href="index.shtml"><img SRC="gene-quantification-logo.gif" ALT="gene.quantification@www.wzw.tum.de" height=135 width=720></a> <p><b><i><font face="Arial,Helvetica"><font size=-1>©  Dr. Michael W. Pfaffl      <a href="mailto:gene.quantification@wzw.tum.de">gene.quantification@wzw.tum.de</a></font></font></i></b></center> <hr ALIGN=LEFT NOSHADE WIDTH="100%"> <center><font face="Arial,Helvetica">Main focus of GENE QUANTIFICATION web page is to describe</font> <br><font face="Arial,Helvetica">and summarize all technical aspects involved in quantitative </font> <br><font face="Arial,Helvetica">gene expression analysis using real-time PCR  &  RT-PCR.</font> <br><i><font face="Arial,Helvetica">( specificity, sensitivity, reproducibility, intra- & inter-assay variations, </font></i> <br><i><font face="Arial,Helvetica">kinetic PCR efficiency calculation, optimization strategy, etc.)</font></i> <p><font face="Arial,Helvetica">The GENE QUANTIFICATION web page illustrates the usefulness of a reliable quantification strategy, and the difference between absolute vs. relative quantification assays in kinetic PCR & kinetic RT-PCR.</font> <p><font face="Arial,Helvetica">RT-PCR is the technique of choice for analyzing mRNA in extremely low abundance. Real-time RT-PCR using SYBR Green I detection combines the ease and necessary exactness to be able to produce reliable as well as rapid results. </font> <br><font face="Arial,Helvetica">To obtain high accuracy and reliability in RT and real-time PCR highly defined </font> <br><font face="Arial,Helvetica">quantification strategies and calculation methods are needed.</font></center> <table WIDTH="100%" > <tr> <td WIDTH="10%"></td> <td> <br><b><font face="Arial,Helvetica"><font size=+2>    <i><u>Gene Quantification content:</u></i></font></font></b> <ul> <li> <font face="Arial,Helvetica"><font size=+1><a href="real-time.html">real-time  PCR  and  RT-PCR </a></font></font></li> <li> <font face="Arial,Helvetica"><font size=+1><a href="mrna.html">mRNA structure  and  overview of mRNA analysis methods</a></font></font></li> <li> <font face="Arial,Helvetica"><font size=+1><a href="optimization.html">Optimization strategies for kinetic RT-PCR</a></font></font></li> <li> <font face="Arial,Helvetica"><font size=+1><a href="platform.html">Available  real-time PCR  platforms</a></font></font></li> <li> <font face="Arial,Helvetica"><font size=+1><a href="block.html">Classical   block RT-PCR</a></font></font></li> <li> <font face="Arial,Helvetica"><font size=+1><a href="strategy.html">Description  of  quantification  strategies  in  real-time RT-PCR</a></font></font></li> <li> <font face="Arial,Helvetica"><font size=+1>Description  of  <a href="absolute.html">absolute quantification</a>  and  <a href="relative.html">relative quantification</a></font></font></li> <li> <font face="Arial,Helvetica"><font size=+2><a href="rest.html"><b>REST ©</b><i>(Relative  Expression  Software  Tool)</i></a></font></font></li> <li> <font face="Arial,Helvetica"><font size=+1><a href="hkg.html">Housekeeping Gene  problematic  <b>( under construction !!! )</b></a></font></font></li> <li> <font face="Arial,Helvetica"><font size=+1><a href="array.html">Verification of array results <b>( under construction !!! )</b></a></font></font></li> <li> <font face="Arial,Helvetica"><font size=+1><a href="competitive.html">Comparison  of   real-time RT-PCR   vs.   competitive RT-PCR</a></font></font></li> <li> <font face="Arial,Helvetica"><font size=+1><a href="download.html">Download  of  software  tools  <b>( <i>REST ©</i> )</b></a></font></font></li> <li> <font face="Arial,Helvetica"><font size=+1><a href="literature.html">Summary  of  relevant  literature</a></font></font></li> <li> <font face="Arial,Helvetica"><font size=+1><a href="link.html">Summary  of  relevant  links</a></font></font></li> <li> <font face="Arial,Helvetica"><font size=+1><a href="link.html">Newsgroups  &  Biotech Boards</a></font></font></li> </ul> </td> </tr> <tr> <td></td> <td></td> </tr> </table> <center> <p><font face="Arial,Helvetica">The reverse transcription - polymerase chain reaction (RT-PCR) is the most sensitive method for the detection of low abundance mRNA, often obtained from limited tissue samples. However, real-time RT-PCR is a complex technique, there are substantial problems associated with its true sensitivity, reproducibility and specificity and, as a quantitative method, it suffers from the problems inherent in RT and PCR. The recent introduction of fluorescence based kinetic (real-time) RT-PCR procedures significantly simplifies the process of producing reproducible quantification of mRNAs and promises to overcome these limitations. Nevertheless, their successful application depends on a clear understanding of the practical problems, and careful experimental design, application and validation remain essential for accurate quantitative measurements of the transcriptom.</font></center> </td> </tr> <tr> <td></td> <td> <center><i><font face="Arial,Helvetica"><font size=-1>© <a href="mailto:gene.quantification@wzw.tum.de">gene.quantification@wzw.tum.de</a></font></font></i> <p><font face="Arial,Helvetica"><font size=-2>optimized for <b>Netscape Navigator v4.7</b> and for <b>Internet Explorer v5.0</b> (and higher)</font></font> <br><font face="Arial,Helvetica"><font size=-2>optimal resolution 1024 x 768 pixel</font></font></center> </td> </tr> </table> <br>  </body>