TaqMan miRNA Assay

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