TaqMan miRNA Assay

Revision as of 18:52, 30 December 2014 by Davebridges (Talk | contribs) (wrote first half)

(diff) ← Older revision | Latest revision (diff) | Newer revision → (diff)
Revision as of 18:52, 30 December 2014 by Davebridges (Talk | contribs) (wrote first half)

(diff) ← Older revision | Latest revision (diff) | Newer revision → (diff)


Materials

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