Blood mirna kit

Total RNA was extracted from blood samples collected from children using the Blood miRNA Kit (PreAnalytiX, Hombrechtikon, Switzerland). The purity and quantity of total RNA were evaluated using the ND-1000 Spectrophotometer (NanoDrop, Wilmington, NC, USA). Absorbance measurements at 260 nm are commonly used to quantify RNA. The RNA sample was considered to be relatively pure if the ratio of absorbance at 260 nm to absorbance at 280 nm or 230 nm was ranged from 1.7 to 2.0. RNA integrity was evaluated based on an RNA Integrity Number (RIN) value greater than or equal to 8 using the Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA, USA).

Whole blood from all patients and healthy controls was collected in PAXgene Blood RNA Tubes according to the manufacturer’s protocol and saved at -20 °C. RNA was extracted using PAXgene Blood miRNA kit (PreAnalytiX, Switzerland). Sample quality was assessed using Agilent Technologies Bioanalyzer with Agilent RNA 6000 Nano Kit (Agilent Technologies, Sweden). RNA-seq was based on the Illumina HiSeq 2000 platform and TruSeq library construction, with 20-m pair-end reads per sample (Illumina, CA, USA). For the RNA-seq data from the initial discovery cohort and the COMBINE validation cohort, TopHat-Cufflinks 2.0 software package was used for RNA-seq data alignment and analysis, as described previously [14 (link)], against an hg19 USCS human genome reference. Cufflinks 2.0 was used for abundance estimation and quantification of transcripts. These data have been deposited in the NCBI Gene Expression Omnibus repository [GEO:GSE90081] [15 ]. The PBMC samples for the COMBINE validation cohort were collected as citrated blood and isolated using Ficoll-Paque (GE Healthcare, Sweden). A detailed description of the validation cohort has been published previously [16 ].

Prior to RNA extraction, frozen whole-blood PAXgene samples were thawed at room temperature for 2 h and subjected to RNA extraction using PAXgene Blood miRNA Kit (PreAnalytix/QIAGEN, Cat# 763134). Total RNA, including RNA longer than approximately 18 nucleotides, was purified, following the protocol supplied by the kit manufacturer. Sample concentration was measured with Nanodrop 2000 spectrophotometer and Qubit Fluorometric Quantitation (Thermo Fisher Scientific). The quality of the samples was ensured with Experion Automated Electrophoresis System (Bio Rad) and Agilent 2100 Bioanalyzer RNA Pico chip. Library preparation and sequencing were carried out at the Finnish Functional Genomics Centre (FFGC). Before starting library preparation, ERCC Spike-in Mix 1 (Invitrogen P/N 4456739) was added to 100 ng RNA according to the kit's protocol. RNA-seq libraries were prepared using TruSeq stranded mRNA HT kit and protocol # 15031047 (Illumina). The quality and quantity of the amplified libraries were measured using Advanced Analytical Fragment Analyzer (Agilent) and Qubit Fluorometric Quantitation, respectively. Pooled libraries were sequenced on an Illumina NovaSeq 6000 instrument, using 2 × 50 bp paired-end sequencing.