I'm looking for a good protocol for spike-in for rat serum miRNA quantification. I've heard that the C.elegans spike-in works well, but don't have an appropriate protocol for it. Any tips would be welcome!
Additionally, from all I've read, Agilent Bioanalyzer works better than NanoDrop for serum. True/not true?
We do the same. Cel-39 is spiked in as there is no proper normalization control for miRNA in serum
Thanks a bunch! I've been using Tomasetti et al 2012 and Mitchell et al 2008 for methods using cel-miR-39. Glad to see that's what you are using as well Jochen and Lawrence.
Hi Anne,
I work the same way as Jochen. It works well!
I also used internal controls like U6 and Y1. I found out that they are expressed in similar levels and you can detect them also in serum and urine from rats. Next step is to check if miR-16 is useful as a control. Maybe anybody has already experience?
May I ask what's your subject you're working on?
Hi Anne,
Pls see this paper by Turchinovich et al., 2011, Nucleic Acids Research.
doi:10.1093/nar/gkr254
Also the miRNeasy Serum/Plasma Kit from Qiagen has a synthetic cel-miR-39 as Spike-In Control incorporated in the kit.
I am posting in the same thread as discussion here is active and I am facing the same problem.
I am following the same protocol as given by miRNeasy Serum plasma kit with 10 times Qiazol than starting serum sample(Mitchell PS). But somehow I feel my cel mir 39 is getting degraded and I am getting Ct of 23-24. My target markers are giving expected Ct but I have been facing the problems with spike ins. I am reconstituting cel mir 39 using directions from Qiagen kit only and adding the same amounts as directed.
Anne did you get try anything with spike in?
Help with troubleshooting will be very much appreciated.
Thanks,
Minakshi
Thanks so much for your input! It is greatly appreciated and I'll take it all under advisement :)
Minakshi,
I haven't run my samples yet. I wanted to see if the cel spike in was what most recommended before going ahead, as I don't have a tonne of sample. I'll let you know how it turns out.
Is it at all possible to add the cel-miR-39 to your sample after RNA isolation? Will that work as a normalizer if I have old samples that I want to validate but never added the spike-in with the Trizol?
We published a good protocol in J Clin Med. 2015 Dec; 4(12): 2012–2027.
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