https://bridgeslab.sph.umich.edu/protocols/index.php?action=history&feed=atom&title=TaqMan_miRNA_Assay 2026-05-30T23:47:56Z Revision history for this page on the wiki MediaWiki 1.45.1 https://bridgeslab.sph.umich.edu/protocols/index.php?title=TaqMan_miRNA_Assay&diff=886&oldid=prev 2014-12-30T18:52:52Z

<p>wrote first half</p> <p><b>New page</b></p><div>[[ Category: miRNA ]]<br /> [[ Category: Gene Expression ]]<br /> [[ Category: qPCR ]]<br /> <br /> ==Materials==<br /> * miRNA template, purified via miRNA kit or from [[ Purification of miRNA and mRNA with TRIzol‎ ]]. Need 1-10 ng per reaction<br /> * Reverse Transcriptase Kit <br /> * TaqMan Assay Kit for both the control and the miRNA of interest<br /> <br /> The general outline is to make a probe-specific cDNA (as opposed to mRNA qPCR where the same cDNA is used for several probes) then to quantify this cDNA. At a minimum you need two probes, U6 (or another control) and your miRNA of interest. To order the TaqMan kit for a particular miRNA go to http://www.lifetechnologies.com/us/en/home/life-science/pcr/real-time-pcr/real-time-pcr-assays.html<br /> <br /> ==cDNA Synthesis==<br /> * Prepare master mix, enough for each RT reaction (need one for each primer/template set). The following volumes are per reaction<br /> ** 0.15 uL 100 mM dNTPs<br /> ** 1 uL MultiScribe Reverse Transcriptase<br /> ** 1.5 uL 10X Reverse Transcriptase Buffer<br /> ** 1.9 uL RNAse Inhibitor<br /> ** 4.16 uL Nuclease Free Water <br /> * You will then prepare a tube for each primer/RNA set as such in a PCR tube:<br /> ** 7 uL master mix from above<br /> ** 5 uL total RNA (presumes this is 1-10 ng)<br /> ** 3 uL of 5X RT primer from the appropriate TaqMan kit<br /> * Incubate on ice for 5 minutes, and keep on ice until ready to start the assay<br /> * Amplify the cDNA in a thermal cycler with the following program (TaqMan cDNA Synthesis):<br /> ** 16C for 30 min<br /> ** 42C for 30 min<br /> ** 85C for 5 min<br /> ** 4C hold<br /> <br /> ==qPCR Reaction==</div>

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