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Preparation of highly multiplexed small RNA sequencing libraries

Persson, Helena LU ; Søkilde, Rolf LU ; Pirona, Anna Chiara and Rovira, Carlos LU (2017) In BioTechniques 63(2). p.57-64
Abstract

MicroRNAs (miRNAs) are ~22-nucleotide-long small non-coding RNAs that regulate the expression of protein-coding genes by base pairing to partially complementary target sites, preferentially located in the 3´ untranslated region (UTR) of target mRNAs. The expression and function of miRNAs have been extensively studied in human disease, as well as the possibility of using these molecules as biomarkers for prognostication and treatment guidance. To identify and validate miRNAs as biomarkers, their expression must be screened in large collections of patient samples. Here, we develop a scalable protocol for the rapid and economical preparation of a large number of small RNA sequencing libraries using dual indexing for multiplexing. Combined... (More)

MicroRNAs (miRNAs) are ~22-nucleotide-long small non-coding RNAs that regulate the expression of protein-coding genes by base pairing to partially complementary target sites, preferentially located in the 3´ untranslated region (UTR) of target mRNAs. The expression and function of miRNAs have been extensively studied in human disease, as well as the possibility of using these molecules as biomarkers for prognostication and treatment guidance. To identify and validate miRNAs as biomarkers, their expression must be screened in large collections of patient samples. Here, we develop a scalable protocol for the rapid and economical preparation of a large number of small RNA sequencing libraries using dual indexing for multiplexing. Combined with the use of off-the-shelf reagents, more samples can be sequenced simultaneously on large-scale sequencing platforms at a considerably lower cost per sample. Sample preparation is simplified by pooling libraries prior to gel purification, which allows for the selection of a narrow size range while minimizing sample variation. A comparison with publicly available data from benchmarking of miRNA analysis platforms showed that this method captures absolute and differential expression as effectively as commercially available alternatives.

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author
organization
publishing date
type
Contribution to journal
publication status
published
subject
keywords
Biomarker, MicroRNA, Non-coding RNA, Sequencing
in
BioTechniques
volume
63
issue
2
pages
8 pages
publisher
Informa Healthcare
external identifiers
  • scopus:85027304620
  • wos:000407562400003
ISSN
0736-6205
DOI
10.2144/000114574
language
English
LU publication?
yes
id
59aabd13-cb2b-48fe-9579-cd0d1c819341
date added to LUP
2017-09-01 14:23:20
date last changed
2017-09-18 11:43:59
@article{59aabd13-cb2b-48fe-9579-cd0d1c819341,
  abstract     = {<p>MicroRNAs (miRNAs) are ~22-nucleotide-long small non-coding RNAs that regulate the expression of protein-coding genes by base pairing to partially complementary target sites, preferentially located in the 3´ untranslated region (UTR) of target mRNAs. The expression and function of miRNAs have been extensively studied in human disease, as well as the possibility of using these molecules as biomarkers for prognostication and treatment guidance. To identify and validate miRNAs as biomarkers, their expression must be screened in large collections of patient samples. Here, we develop a scalable protocol for the rapid and economical preparation of a large number of small RNA sequencing libraries using dual indexing for multiplexing. Combined with the use of off-the-shelf reagents, more samples can be sequenced simultaneously on large-scale sequencing platforms at a considerably lower cost per sample. Sample preparation is simplified by pooling libraries prior to gel purification, which allows for the selection of a narrow size range while minimizing sample variation. A comparison with publicly available data from benchmarking of miRNA analysis platforms showed that this method captures absolute and differential expression as effectively as commercially available alternatives.</p>},
  author       = {Persson, Helena and Søkilde, Rolf and Pirona, Anna Chiara  and Rovira, Carlos},
  issn         = {0736-6205},
  keyword      = {Biomarker,MicroRNA,Non-coding RNA,Sequencing},
  language     = {eng},
  number       = {2},
  pages        = {57--64},
  publisher    = {Informa Healthcare},
  series       = {BioTechniques},
  title        = {Preparation of highly multiplexed small RNA sequencing libraries},
  url          = {http://dx.doi.org/10.2144/000114574},
  volume       = {63},
  year         = {2017},
}