Hello Sneha

I have study total RNA extraction from LDL isolated of human plasma. In order to study miRNA expression. RNA concentration obtained was 11 - 15 ng/uL with NucleoSpin miRNA plasma Kit (Macherey-Nagel). The PCR was OK.

According to Taqman miRNA assay the concentration is 10ng.

I advice you check the sensibility of fragment analyzer to determinate the sample concentration. In addition, you could increase your sample volume using some reference molecule as LDL concentration for your Qiagen miRneasy kit

Best regards.

You will want to load as much miRNA into the cDNA synthesis kit as you can.

The cDNA kits will have a maximum volume for this, thus limiting how much you can volume input from plasma.  For example, if your cDNA synthesis kit has a 10uL max volume, your maximum cDNA input will be .607ng X 10 = 1.6 ng total.

I worked with tissue miRNA using 500-1000ng input which worked fine for high to middle-abundance miRNAs. I think you may have hard time making quantitative statements (X-fold change) and the best you could do is maybe qualitative statement (is miRNA X present in my serum/plasma, yes or no). However even this may be difficult because your input is so low that you do not now if a sample was negative for a miRNA beause of low input or was it actually caused by a treatment effect?  As mentioned above by Oscar, consult the kit to determine the limits of your assay. 

Thank you very much for your inputs. I have been using endogenous controls like miR-423-5p and miR-451a studied to be good reference controls in serum and plasma. I have also used an exogenous control i.e. ath-miR-159a to account for the variability. Even with an extremely low concentration of RNA in serum and plasma, the TaqMan miR assay was still able to detect miRNAs in my healthy controls with plasma yielding a better Ct value. 

I am confused if I have to perform any fold change studies even in my healthy controls? If so, is there a target miRNA that is stably expressed in healthy controls? Literature proposes that there is no single miRNA that is stably expressed in serum and plasma. I will perform a similar study with my patient samples for which I can use a miR target and determine the diferential expression between healthy and patient samples. Any inputs or suggestions on this would be helpful!

Thanks,

Is the 11-15ng RNA /uL serum the total RNAs, not just miRNAs?

Thanks,

Jamie

Hello, Jamie,

We have a high sensitivity RNA fragment analyzer that gives us the distribution of the total RNA profile at our core facility. The amount mentioned above was the total RNA content per microliter volume.

Thanks Sneha,

Would that be human or mouse serum?

It was human serum. Serum from healthy volunteers to be more specific.

"Literature proposes that there is no single miRNA that is stably expressed in serum and plasma."

Is that based on a genomic analysis i.e. via microarray/NGS? I found it somewhat difficult to believe there is no miRNA that is stably expressed, though it is possible. If you cannot find a stable gene, then you cannot use relative expression, you may have to use a standard curve or digital PCR.

Hi Sneha, What is the usual yield (ng) of your total RNA/miRNA per ml of plasma?

In my hands (using miRneasy, too) I am getting up to 1ug total RNA/mL of plasma, most of them in the exosome fraction