Hi Juliana, I have succesfully prepared libraries with the TruSeq and small RNA Sample Preparation Kit with RNA from around 300-800ul mouse serum. I get around 500ng/ml of RNA from mouse serum (quantified by Qubit Fluorometer). Human serum/plasma tend to have a lower concentration of RNA than mouse. I would extract RNA from as much plasma you can get and pre-concentrate it (speed-vac). Hope it helps.

But what extraction protocol are you using? because I'm following instructions from miRNeasy plasma kit, and I've achieved 40-60pg/uL on Bioanalyzer (with 200uL as starting material). Then I tried to extract from 1mL plasma but the concentration was similar...

Thank you, Anna, for your help.

Hi Juliana, I don't think Bioanalyser is the best for quantification of RNA, at least I'm my hands it shows completely untrue values. As a test, run a known concentration of any RNA synthetic ( try maybe a range of concentrations- 20ng/ml, 100ng/ml, 500ng/ml) and see if this is true for you too. And maybe try the same with nano-drop. I know that the quantification limits are not brilliant. I extract with miRVANA Paris, but I know people also use the Qiagen kit for other liquid samples and work fine. For quantification I use Qubit-Invitrogen.

Thank you all! I'll try to quantify with Qubit and Nanodrop and pre-concentrate it.

I tested some samples on Nanodrop and the purity was very low. I would like to know if you modified any steps in the extraction protocol to achieve better results on quantification and purity quality.

We don't prepare microRNAs from serum or plasma, but total RNA (of which only a fraction is microRNA). For mouse and human serum/plasma, we start with 100 or 200 µl, respectively (no linear benefit of using more). We typically get yields in the 5-25 ng/µl range (in 14 µl eluate using the micro columns from the Qiagen miRNeasy Serum/Plasma Kit). Yield and concentration are variable, e.g. serum from cancer patient or tumor bearing mouse has much higher circulating RNA concentration. More information on our microRNA gene expression services in the link below.

http://www.biogazelle.com/lab-services/applications/mirna

Hi Jo,

I'm obtaining the same total RNA as you from serum using mirVana Paris kit but when I measure in Qubit 1.0 the RNA is not detected. I wonder if this concentration can be only with DNA contamination (I really hope not). Can I trust Nanodrop for NGS applications once Qubit can't measure my RNA samples?

Oh no, the total RNA we isolate is devoid of DNA.

Hi Jo Vandesompele,

I have also isolated total RNA with the Qiagen miRNeasy Serum/Plasma kit. Only when I measure the concentration with the Qubit I get stock concentrations ranging from 0.57 - 5.21 ng/ul. I was wondering whether you changed something in the protocol of Qiagen? And did you als make cDNA from your samples? Because I would like to use Taqman for making cDNA of a specific miRNA, but the protocol uses total RNA as starting material, but I did not measure total RNA, and it appears that the Nanodrop is very inaccurate at these low concentrations (so I am unable to measure total RNA). How did you manage that problem? Did you do a dilution series with the total RNA you have isolated?

Hey together,

does anyone of you tried this kit using bacterial RNA?