miRNA Sequencing with RNAstable® and SeqMatic miRNA kit

Sample Information

  • <10ng of human miRNA (small ~16nt RNA) was extracted from samples using Qiagen miRNeasy® extraction kit.
  • 12 samples were stored using RNAstable® with standard protocol
  • Samples were shipped from South America to the US via a standard package envelope

 


Sample Processing

  • Samples were reconstituted with nuclease free water.
  • Samples sent directly to library generation without cleanup.
  • Sequencing libraries were generated using the SeqMatic miRNA library preparation kit.

 


Library Generation Overview

  • Unique 3’ and 5’ adapter oligos were ligated to the ends of each miRNA.
  • cDNA was generated, followed by PCR.
  • A unique barcode was added for multiplexed sequencing.

 


Library Quality

  • RNAstable samples look identical to control RNA.
  • High yield: samples generated > 2nM of library necessary for sequencing.
  • High purity: clean separation of adapter dimer bands after gel electrophoresis.

 


Sequencing

  • Sequencing was performed on the Illumina® MiSeq® instrument.
  • A single read of 50bp is sufficient for miRNA profiling and expression analyses.
  • Instrument provides a comprehensive sequencing platform with onboard clustering, sequencing, and data analysis.

 


Data Analysis

  • All libraries aligned to human miRbase.
  • Adapter dimer contamination rate was very low.
  • Replicate samples generated similar expression profiles with high R2 value.

 


Summary

  • RNAstable provides a good method of shipping low amounts of miRNA across the world.
  • RNAstable does not interfere with enzymatic reactions during library generation.
  • SeqMatic miRNA sample preparation kit provides a robust method of generating Illumina sequencing libraries with very low sample input.
  • Libraries generated showed no loss of yield or any biases.
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