View source for TaqMan miRNA Assay

[[ Category: miRNA ]]
[[ Category: Gene Expression ]]
[[ Category: qPCR ]]

==Materials==
* miRNA template, purified via miRNA kit or from [[ Purification of miRNA and mRNA with TRIzol‎ ]].  Need 1-10 ng per reaction
* Reverse Transcriptase Kit 
* TaqMan Assay Kit for both the control and the miRNA of interest

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

==cDNA Synthesis==
* Prepare master mix, enough for each RT reaction (need one for each primer/template set).  The following volumes are per reaction
** 0.15 uL 100 mM dNTPs
** 1 uL MultiScribe Reverse Transcriptase
** 1.5 uL 10X Reverse Transcriptase Buffer
** 1.9 uL RNAse Inhibitor
** 4.16 uL Nuclease Free Water 
*  You will then prepare a tube for each primer/RNA set as such in a PCR tube:
** 7 uL master mix from above
** 5 uL total RNA (presumes this is 1-10 ng)
** 3 uL of 5X RT primer from the appropriate TaqMan kit
* Incubate on ice for 5 minutes, and keep on ice until ready to start the assay
* Amplify the cDNA in a thermal cycler with the following program (TaqMan cDNA Synthesis):
** 16C for 30 min
** 42C for 30 min
** 85C for 5 min
** 4C hold

==qPCR Reaction==