Preparation of highly multiplexed small RNA sequencing libraries

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Preparation of highly multiplexed small RNA sequencing libraries. / Persson, Helena; Søkilde, Rolf; Pirona, Anna Chiara ; Rovira, Carlos.

In: BioTechniques, Vol. 63, No. 2, 2017, p. 57-64.

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TY - JOUR

T1 - Preparation of highly multiplexed small RNA sequencing libraries

AU - Persson, Helena

AU - Søkilde, Rolf

AU - Pirona, Anna Chiara

AU - Rovira, Carlos

PY - 2017

Y1 - 2017

N2 - 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.

AB - 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.

KW - Biomarker

KW - MicroRNA

KW - Non-coding RNA

KW - Sequencing

U2 - 10.2144/000114574

DO - 10.2144/000114574

M3 - Article

VL - 63

SP - 57

EP - 64

JO - BioTechniques

T2 - BioTechniques

JF - BioTechniques

SN - 0736-6205

IS - 2

ER -