There are several methods to study miRNAs from blood samples and most commnly PCR. To compare the concentration we use deltaCt as their unit to compare references. But I am wondering what is the expected range of miRNA concentration in Molars in the blood. Is there any comperative study?
Hi Osman
The range of concentrations of miRNAs in blood vary from nanomolar to femtomolar values. In terms of Ct values, you can expect something between 20 and 35 units, depending how you perform your cDNA dilutions.
Best luck. Paco
Hi Osman
The range of concentrations of miRNAs in blood vary from nanomolar to femtomolar values. In terms of Ct values, you can expect something between 20 and 35 units, depending how you perform your cDNA dilutions.
Best luck. Paco
I am not convinced that a proper answer exists. Firstly, there is too little miRNA to be quantified directly. For example, in 10 micrograms of brain total RNA (very little RNase activity, so no smear), I can visualise 1-2 nanograms of miRNAs using SYBR Gold staining. In blood, the quantities are even lower, the RNA is degraded and the miRNAs may be found in exosomes or similar structure, rendering a molar expression inaccurate.
To do a direct quantification, one should use dilution curves of synthetic RNA in conjunction with RT-qPCR. Delta Ct by itself assumes perfect priming, perfect logarithmic amplification etc, and does not satisfy the miQE standards that should be met for reliability and publication. Moreover, while qPCR is usually fairly robust, RT is far from it. Try it yourself - input half and double the amount of RNA into your RT reaction and see if you get 1 Ct difference at the end!
I wish I could be more helpful!
Thank you all! I am trying to make a positive control for my experiment in which I use blood serum. So I decided to add synthetic miRNA into healthy blood sample before isolation but I was having trouble how much miRNA should be considered normal. I will start with 1 nM and go down from there.
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