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Sample GSM1112882

Query DataSets for GSM1112882

Status

Public on May 15, 2013

Title

microRNA from sample C constructed by the kit from NEB [LW_CN_2]

Sample type

SRA

Source name

microRNA from sample C constructed by the kit from NEB

Organism

Homo sapiens

Characteristics

tissue: blood
fraction: exosome

Extracted molecule

total RNA

Extraction protocol

we selected plasma samples (samples A, B and C) from 3 anonymous blood donors and split each sample into two for technical replication.
We tested the six samples (A1 and A2, B1 and B2, C1 and C2) using two small RNA library preparation kits: NEBNext Multiplex Small RNA library Prep Set from New England Biolab (NEB) and NEXTflex Small RNA Sequencing Kit from Bioo Scientific (BS). We also tested Illumina’s TrueSeq Small RNA Sample Preparation Kit (ILMN) in sample A1 and A2. Together, we tested these plasma samples by sequencing 14 indexed libraries.
Human plasma samples were obtained from Mayo Clinic and stored at -80°C before use. Exosomes were isolated from 250 µL of plasma prepared from whole blood using the ExoQuick Exosome Precipitation Solution (SBI) according to the manufacturer’s instructions with minor modifications. Briefly, the plasma was incubated with Thromboplastin D (Thermo) for 15 min at 37oC. After centrifugation at 10,000 rpm for 5 min, the supernatant was mixed with 75 µL of ExoQuick Solution and RNase A (Sigma) at final concentration of 10 µg/mL. The mixture was kept at 4oC overnight and then further mixed with 150 units/mL of murine RNase inhibitor (NEB) before centrifugation at 1500 g for 30 min. The exosome pellet was dissolved in 25 µL 1×PBS, 2 µL of which was reserved for evaluation of exosome size and concentration using NanoSight LM 10 instrument (Particle Characterization Laboratories). The remainder was subjected to RNA extraction immediately. The usage of human biospecimen for this study was approved by Institutional Review Board of Medical College of Wisconsin and Mayo Clinic.
RNA was prepared by using miRNeasy Micro Kit (QIAGEN). Twenty-three µL of exosome suspension was mixed with 700 µL QIAzol Lysis buffer, and was processed by the standard protocol from the manufacturer. The extracted RNA was eluted with 14 µL of RNase-free water. The quantity and quality of the RNA were determined by Agilent Bioanalyzer 2100 with Small RNA Chip and RNA 6000 Pico Kit as indicated (Agilent Technologies).
The concentration and the size distribution of isolated exosomes were measured using NanoSight. Prior to sampling, the sample solutions were homogenized by vortexing, followed by serial dilution to the final dilution of 1:100,000 in 0.2 µm-filtered 1x PBS. NIST (National Institute of Standards and Technology) traceable 97 nm ± 3 nm polystyrene latex standards were added and analyzed in conjunction with the diluted exosome solution to validate the operation of the instrumentation. A blank 0.2 µm-filtered 1x PBS sample was also run as a negative control. Each sample analysis was conducted for 90 seconds. The Nanosight’s automatic analysis settings (High Sensitivity, Blue laser [405 nm, 645 mW]) were utilized for the processing of the data. All samples were evaluated in triplicates.

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

size fractionation

Instrument model

Illumina HiSeq 2000

Description

Characterization of human plasma-derived exosomal RNAs

Data processing

Perl Scripts were developed to process data from RNA sequencing. Raw reads were firstly extracted from FASTQ files, and trimmed with sequencing quality control of Q > 13 , and then cleaned up 3’ adaptor sequences within read sequences. Those prepared sequences filtered with the length ≥16 nt were combined into a FASTA file and aligned against human miRNA sequences downloaded from miRBase (Release 19, 2043 entries) and human genome reference sequences downloaded from NCBI ftp site (Release 103) with Bowtie (version 0.12.8) , of which the parameters for alignment were -m 3 -n 1 -f -a -best --strata. Normalization of miRNA profiling was the read counts of a target miRNA by million bases versus the sum of read counts of all mappable miRNAs on human miRNA sequences.
Genome_build: miRNA from miRBase(release19)
Supplementary_files_format_and_content: Data frame of normalized miRNA transcription level in *.txt

Submission date

Apr 03, 2013

Last update date

May 15, 2019

Contact name

Liang Wang

E-mail(s)

liwang@mcw.edu

Phone

414-955-2574

Organization name

Medical College of Wisconsin