NGI during Covid-19 outbreak

NGI is still up and running during the Covid-19 pandemic, but we are experiencing some limitations in terms of personnel and key reagents. Each NGI node is following its respective host university recommendations and will continue operation until further notice.

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QIAseq miRNA low input

Generation of miRNA libraries from very low input total RNA samples and degraded total RNA.

QIAseq miRNA Library kit is specifically designed for very low input total RNA samples. It also works with degraded total RNA. Please check the sample requirements and expected results below.

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%)
  • Sample origin: plasma/serum, LCM, FFPE, cell-free, sorted cells, biopsies
  • 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 to use:

  • 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

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 able to generate a library from it.

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. Up to a maximum of 48 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 checking 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

Bioinformatics

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

Applications
Relevant Technologies
Bioinformatics Pipelines
Method Status

Pilot

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

Keywords