I use TaqMan so will not comment of your question about standard curve as Taqman chemistry only needs verification of amplification efficiency for each probe (to achieve relative expression values between controls and disease). However, I have used cel-miR-39 following way.
Prepare carrier/spike RNA solution (15x master):
10.5 μl MS2 RNA
75 μl of 5fmol/μl cel-mir-39 (total 375 fmol).
Use 5.7 μl of this solution per extraction which equals to 25 fmol. The MS2 RNA increases the consistency of miRNA extraction by working as a carrier. I have used Norgen Total RNA column purification kit but this should work the same way for any column purification system. The important thing is that you add the carrier/spike solution (e.g. cel-mir-39) AFTER adding the "lysis solution" to prevent the degradation by RNAses.
Hope this helps,
JanneMember Since 12 Feb 2010
Offline Last Active Feb 11 2013 12:40 AM
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ALS, RNA oxidation, mitochondria