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Old 09-13-2013, 05:28 AM   #1
lran2008
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Default miRNA qPCR normalization by spike-in miRNA

Hi All,

There are some commonly used reference genes for normalization of miRNA qPCR, like 5S, 18S rRNA and U6 snRNA. But they might not be the right one in a specific tissue. So would spike-in miRNA perform better for normalization?
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Old 09-13-2013, 05:37 AM   #2
DrOM
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The aims for normalization of qRT-PCR is to normalize for fluctuations in input either quantitatively or qualitatively among others. If you are using a spike in miRNA you cannot control for this. Therefore it is not useful in my eyes.
I would suggest to run a test with a subset of samples and 3-5 different housekeepers and then run algorithms like these here
http://www.leonxie.com/referencegene.php?type=reference

to investigate the variance between samples. Then choose those with least variance between samples for that tissue.
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Old 09-13-2013, 08:09 AM   #3
lran2008
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Quote:
Originally Posted by DrOM View Post
The aims for normalization of qRT-PCR is to normalize for fluctuations in input either quantitatively or qualitatively among others. If you are using a spike in miRNA you cannot control for this. Therefore it is not useful in my eyes.
I would suggest to run a test with a subset of samples and 3-5 different housekeepers and then run algorithms like these here
http://www.leonxie.com/referencegene.php?type=reference

to investigate the variance between samples. Then choose those with least variance between samples for that tissue.
There are some studies which actually spike-in miRNA for normalization.
eg. http://onlinelibrary.wiley.com/doi/1...ijc.25376/full

I am a beginner, so could you point out the reason why this is not useful?
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Old 09-13-2013, 08:24 AM   #4
DrOM
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Quote:
Originally Posted by lran2008 View Post
There are some studies which actually spike-in miRNA for normalization.
eg. http://onlinelibrary.wiley.com/doi/1...ijc.25376/full

I am a beginner, so could you point out the reason why this is not useful?
In the quoted paper they use the spike in-controls (cel-miRNAs) to monitor for isolation efficiency of RNA from circulating tumor cells. Due to minute amounts they could not detect or measure the total RNA from these samples. So maybe you should first describe your experiment more thoroughly? If you want to detect miRNAs by qRT-PCR from total RNA, which concentration you can measure after isolation, spike-in controls do not make much sense, since isolation procedures are pretty standard for miRNAs including total RNA. One could use a miRNA, e.g. miR-16, to control if the method looses the miRNAs, but I surely would use cell lines for the establishment of this method in your lab.
By the way I am not sure if this is the right forum for this question, or do you also plan to sequence miRNA libraries?
Hope this helps

Last edited by DrOM; 09-13-2013 at 08:26 AM.
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