Difference between revisions of "TaqMan miRNA Assay"
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Latest revision as of 18:52, 30 December 2014
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
