QIAseq miRNA

miRNA libraries from very low input total RNA samples & degraded total RNA.

The QIAseq miRNA Library kit is specifically designed for low-input total RNA samples. It also works with degraded total RNA. Specially designed for biofluids. Upon request, get access to RAP – RNA Analysis Portal to perform your own data analysis.

Sample requirements

  • Sample type: Total RNA
  • Sample amount:
    • Range: 1-500 ng in 8 μl
    • Recommended: 10 ng in 8 μl
  • Sample volume: 12 μl (4 μl used for Reception Control and 8 μl for library prep)
  • Sample quality: high quality or degraded (DV200 >20%), when working with total RNA.
  • Sample origin: plasma/serum, LCM, FFPE, cell-free, sorted cells, biopsies
    • When working with exosome samples prepared from serum and plasma samples, the recommended starting amount of total RNA is 5 µl of the RNA eluate when 1 ml of serum/plasma has been processed using the exoRNeasy Kits. The original sample should not have signs of hemolysis.
  • Sample extraction method: we recommend using a column-based kit without Phenol.
    • The samples should be resuspended in nuclease-free water
    • Double-check that the extraction kit retains small RNAs (<200bp)
    • Samples should not contain gDNA.
      • We recommend not to overload the extraction kit to avoid gDNA presence.
      • gDNA seems to inhibit enzymatic reactions used for library prep when abundant.
      • Be sure that the total RNA extraction kit removes gDNA.
  • IMPORTANT: The kit specifically ligates the adapter to both the 3’ end and 5’ end of RNAs enabling universal reverse-transcription and library preparation of mature miRNAs, while minimizing the background from other RNA species. This will happen to all RNA species with a 3’ hydroxyl group and a 5’ phosphate group.

How to evaluate the sample quality

Even when we perform a Reception Control in all samples, we require from our users to asses both total RNA quality and concentration.
To accomplish this we recommend using the following:

  • RNA concentration
    • Fluorometric measurements (Qubit, Quant-it)
    • Do not use absorbance measurements (Nanodrop, spectrophotometer)
  • RNA quality
    • Capillary electrophoresis (Fragment Analyzer, Bioanalyzer, TapeStation)
    • Do not use gel electrophoresis
  • RNA quality for FFPE samples
    • Use capillary electrophoresis to determine DV200
  • QC qPCR: useful for samples with low RNA content such as serum, plasma, urine, and other biofluids (e.g.: this kit).
    • Spike-in controls within qPCR can assist in monitoring reproducibility between miRNA isolations, detect the presence of enzymatic inhibitors and nucleases, assess sample integrity for hemolysis (important for serum and plasma miRNA identification), and conduct thorough QC of NGS data by evaluating the reproducibility and linearity of reads mapped to exogenous sequences.

IMPORTANT: If your sample cannot be assessed using the recommended methods or if it is below the range of detection, please contact us, perhaps, we will still be able to generate a library from it.
Qubit HS RNA, Bioanalyzer Pico RNA, and Fragment Analyzer HS RNA are recommended solely for samples containing total RNA (where tRNA and/or rRNA are present).

What we do with your samples

Once your samples arrive at NGI, we start by performing a Reception Control step in which we make sure the sample meets our requirements.
If the samples fail this Quality Control step, we will contact you to discuss possible options.
If the samples pass Reception Control, we will inform you and the samples will be queued for Library prep.

Library preparation

Adapters are ligated sequentially to the 3’ and 5’ ends of miRNAs. The ligation process is dependent on 3’OH and 5’PO4, respectively. Due to this, any RNA molecule with less than 30 bases in length, with the available phosphate and hydroxyl groups, will be part of the library, even degraded RNA. Then, the following steps include: universal cDNA synthesis with the incorporation of UMIs, cDNA cleanup, library amplification, and library cleanup. Bead-based cleanups eliminate library fragments with inserts longer than 30 bases (including mRNAs, RNAs, and others). During data analysis, you can take advantage of the UMIs removing PCR duplicates as well as sequencing artifacts. A maximum of 96 samples can be multiplexed.

Library QC and sequencing

In this step, we evaluate the library yield and determine their size distribution. We will inform you of the QC status of each sample. Once the libraries have passed this QC step, they are queued for sequencing.
The sequencing will be carried out following the setup stated in the agreement.

Expected results

In general, we have been successfully generating libraries that provide enough good quality reads for differential expression analyses. But, the sequencing results from this library prep will be determined by the characteristics of the sample: 

  • A high percentage of PCR duplicates (40% or more) when:
    • The sample input is <10 ng
    • The sample diversity is intrinsically low
    • No worries, these can be identified using the UMIs
  • The yields of the library prep could be insufficient when the input is <1 ng:
    • These samples will probably generate a good quality library but we cannot guarantee a successful preparation
    • if the library concentration is too low, to generate an even pooling is more difficult, so you could expect to have uneven reads yields after sequencing


smrnaseq is a bioinformatics analysis pipeline used for small RNA sequencing data. You will find the details in the nf-core/smrnaseq section.

QIAGEN provides NGI users with access to RAP which stands for RNA Analysis Portal. This cloud-based bioinformatic solution takes your NGS analyses further and provides a fully integrated analysis and biomarker discovery workflow. The best part is, you don’t need any programming skills to use it. It is also wet lab bench-friendly, making it easier for you to use it for your analysis.

Contact us for more info.

Last Updated: 29th April 2024

Please read our sample submission instructions before sending samples:

Sample Submission Guidelines
Relevant Technologies
Bioinformatics Pipelines
Method Status


We are currently testing this method. Please contact us to find out more.