Hi,

1) We usually use the Agilent Bioanalyzer Pico RNA. Anyway, you are not so restricted by integrity number in terms of miRNA... Also you can use the smear concentration analysis (in the size region of miRNAs) to at least normalise the samples between each other before library prep.

2) As more non-target transcripts you have in your library, as deeper you need to sequence to get an appropriate sequencing depth. We usually use separation systems like PippinHT to enrich for miRNA.

3) After the library prep your library will be of adequate size (miRNA + adaptors and barcodes) and adequate concentration (after PCR). So the QC methods are usual - DNA Bioanalyzer and HS Qubit.

Best,

Michail

Hi Ege,

well, that‘ probably because there isn’t the “one” single standardized protocol – there are many ways to skin a cat…

To begin with, here’s a paper that you might find useful: https://academic.oup.com/nar/article/44/13/5995/2457640

Regarding your questions:

1) I don’t think it’s important to know the exact concentration of miRNAs (that’s also pretty hard to quantify), but it certainly doesn’t hurt to know the total RNA concentration. If it’s impossible to accurately quantify total RNA (this might happen if you’re working with serum, extracellular vesicles or other cell-free, low-yield samples), the next best choice would be to normalize to input volume of biofluid instead of input quantity of RNA.

I also agree that RIN doesn’t play that much of a role for miRNAs, as they are much more stable towards degradation than longer species. But keep in mind that RNA degradation produces fragments that might fall into the size range of miRNAs and thus exaggerate the proportion of genuine miRNAs.

2) No, most kits actually use total RNA as starting material.

3) As Michail said, capillary electrophoresis is a great way to check the quality of final libraries as you get a nice reading for fragment length which you can compare to the theoretical size of target fragments. We use the Bioanalyzer High Sensitivity DNA Kit (you might have to dilute libraries for accurate sizing; keep an eye on the DNA concentration range provided in the manual).

In terms of quantifying final libraries (which is also very important), you’ll probably want to go with a qPCR-based method. The main advantage of this approach, compared to something such as Qubit or PicoGreen, is the fact that qPCR only quantifies functional molecules with both adaptors. Imagine a byproduct of library prep that lacks one of the adaptors – this would be quantified by fluorescent assays but wouldn’t be able to amplify during clustering. With qPCR (or dPCR, of course), the assay only captures functional molecules, which should correlate closely to cluster density and, thus, sequencing output.

Good luck,

Dominik

Hi, if you want to know the quality of your total RNA, you need to run the RNA 6000 Nano or Pico assay to obtain a RIN. The small RNA assay can give you a percentage of miRNA. For the concentration, using Nanodrop or Qubit is more reliable than values from Agilent Bioanalyzer.

Most of the commercial library prep kits are starting from total RNA.

The main issue with small RNA libraries is to avoid overcycling during PCR amplification and to have the same or similar number of cycles for each sample.

The library is at the end usually analyzed using the Bioanalyzer High Sensitivity Assay. The concentration should be measured by Qubit or qPCR (library quantification kits).

Thank you all for your input, however since I am basically blind to the whole process unfortunately hence I must ask you some questions.

1) Regarding what Michail Yekelchyk wrote: What exactly is a smear concentration analysis? I have looked online and found nothing.

2) Regarding what Dominik Buschmann wrote: A qPCR based method for detecting concentration of the adaptors, one would have to have both 5' and 3' adaptors in order to achieve this?

3) Regarding what Stefan Bauersachs wrote: Again I am assuming you are refering to totalRNA detection rather than miRNA concentration detection regarding Qubit and Nanodrop being better than Agilent?

Again I appreciate all the comments, it really gave me insight as to what I should actually do.

Hi,

I meant the way of RNA concentration measurement based on the Bioanalyzer - it has (though not always fully accurate) that option. As I remember, it calculates it based on the intensity of ladder peaks (the ladder concentration is known). The smear analysis (referring to PerkinElmer LabChip GX software, for example) is the way to get a value of interest (concentration, average size in bp, etc) over the size range of interest (for example for final library it is 200-1000 bp). It may also give you an idea about the concentration of miRNAs if set to rather small diapason, I guess.

Best,

Michail