most of expression biomarkers at the RNA level appear to be extremely sensitive to variety of everyday exposures: food additives, drugs, flue, etc. I am not sure, that normalized expression is an adequate signature. Best way (in my opinion) is to use ddCt within a single subject/person/patient.

As per my knowledge of the NanoString PAN Cancer gene panel, The NanoString analysis is specifically carried out on its own nCounter software which generates three files (1) Raw data (2) Normalised data (3) Ratio data.

Now from the Ratio data, those DE call is 'YES', are only being considered. Ratio data is generated from Normalised data. So, Normalised data won't be the one for representation. What I am saying its associated with gene expression.

miRNA count which you mentioned, maybe different or more or less same.

So, better consult the Nanostring xpert, they responds quickly in chat or mail.

Regards

Saurabh

Dear Bruna,

nanoString miRNA kit includes 5 mRNA as house keeping genes (HK genes), but If I were you I would not consider to compare 2 types of RNA (i.e. miRNA & mRNA) because they are not comparable at all.

microRNA regulation is quite complex and interfere with mRNA and the feedback between those 2 RNA species is not yet clear.

I think I missed a step in your question, you want to find differentially expressed miRNAs in 2 or more populations (or experimental conditions) by normalizing the counts using hk counts?

Could you give more details on the aim of your experiment if possible?

You can contact me directly by message if you do not want to share details... I will try to help you as much as I can, while I used nano@string also.

best

Thank you all for your answer.

Arnaud Beurdeley I will send you a private message with more details regarding my analysis. However, I can explain a little better what I am trying to do.

We want to know if we can use the absolute count number (raw data) or a normalized "count" number for each miRNA and determine their relative expression (relative to another RNA, either mRNA or specific miRNA). It would be similar to RT-qPCR relative quantification.

At first, we would not be comparing the expression among different groups or populations, only determining if said miRNA presents "more expressed" than the RNA-control determined.