Small RNA Sequencing from Plasma

RNA was isolated from plasma samples using protocol described earlier30 (link). SmallRNA libraries generated using the Ion Total RNA-Seq Kit v2 were processed through emulsion PCR using OneTouch 2 (Life Technologies) and the Ion PGM™ Template OT2-200 Kit, then enriched using the OneTouch ES (all Life Technologies). One sample was loaded per Ion-318^™ chip and sequenced on Ion PGM using the Ion PGM™ Sequencing 200 Kit v2 (all Life Technologies). The aligned BAM files were analyzed using Strand NGS (Strand Life Sciences) and differential miRNA expression was determined by a two-fold change in normalized read number.

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Farr R.J., Januszewski A.S., Joglekar M.V., Liang H., McAulley A.K., Hewitt A.W., Thomas H.E., Loudovaris T., Kay T.W., Jenkins A, & Hardikar A.A. (2015). A comparative analysis of high-throughput platforms for validation of a circulating microRNA signature in diabetic retinopathy. Scientific Reports, 5, 10375.

Publication 2015

Ion Total RNA-Seq Kit v2 OneTouch 2 Ion PGM™ Template OT2-200 Kit OneTouch ES Ion PGM™ Sequencing 200 Kit v2

Chip Emulsion Mirna Plasma Total rna seq

Corresponding Organization :

Other organizations : National Health and Medical Research Council, University of Sydney, Centre for Eye Research Australia, University of Melbourne, St Vincents Institute of Medical Research

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Protocol cited in 1 other protocol

Variable analysis

independent variables

RNA isolation protocol

dependent variables

Differential miRNA expression

control variables

Sample type (plasma)
Library generation method (Ion Total RNA-Seq Kit v2)
Emulsion PCR (OneTouch 2)
Template preparation (Ion PGM™ Template OT2-200 Kit)
Enrichment (OneTouch ES)
Sequencing platform (Ion PGM™ Sequencing 200 Kit v2)
Chip type (Ion-318™ chip)
Bioinformatics analysis (Strand NGS)

controls

No positive or negative controls were explicitly mentioned.

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