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[zammybutt]

Hello Everyone
    I am working on serum microrna profiling in liver cancer as part of my research. However, I am very new to this field and at the moment quite confused about the normalization step for serum microrna. As there is no universal or well defined internal control for serum microrna, I will be using synthetic c-elegans mir-39 from QIAGEN for normalization. However, I dont know how to perform this correctly. I shall be very thankful if anyone experienced in serum microrna profiling and have used synthetic mirnas for normalization could explain the following:

1.  The steps to be performed when using c.elegans miR-39 and the protocol/steps to generate standard curve on real time PCR ( e.g., when generating standard curve, what should be used in place of total RNA (RNA from control or diseased samples))? how to generate standard curve?

2.  Do I have to generate standard curve each time from spiked-in serum samples from control and disease?


Regards
Azeem Butt,

[Janne]

Hi Azeem,

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,
J