Preserved blood rna purification kit

Total RNA was isolated from bovine blood using a Preserved Blood RNA Purification Kit (Norgen Biotek Corp, Thorold, ON, Canada), and the quality of RNA was measured using the 2200 RNA ScreenTape TapeStation System (Agilent Technologies Inc., Cedar Creek, TX, United States) producing RNA integrity (RIN) values ranging from 8.0 to 9.8. To prepare the mRNA cDNA libraries, 1.0 μg of total RNA was used from each sample using the TruSeq RNA Library Preparation kit v2 (Illumina, San Diego, CA, United States). Poly A-containing mRNA was enriched from the total RNA using poly-T oligo attached beads and fragmented for first-strand cDNA synthesis, followed by second-strand synthesis. The ends were repaired, and 3′ end adenylation and adapter ligation were performed for each library. Following these steps, libraries were polymerase chain reaction (PCR) amplified, validated using the Bioanalyzer (Agilent Technologies Inc., Cedar Creek, TX, United States), and finally normalized and pooled. Unique indices were used for all samples, and libraries were pooled and sequenced paired end (2 × 100 bp) on four separate lanes on a HiSeq 4000 platform, and sequencing was performed at McGill University and Genome Quebec Innovation Center (Montreal, QC, Canada). In total, 43 samples were used to generate paired-end sequences, and their raw reads were used for downstream analyses.

Approximately 500 µL of the dehosted sample was added to a PowerBead (0.1 mm glass) tube (Qiagen catalog #13118-50) with 500 µL Lysis Buffer A. Bead-beating was performed on a Vortex Genie 2 with a vortex adapter for 10 min at maximum speed. Total nucleic acids were purified with Plasma/Serum RNA Purification Midi kit (Norgen Biotek, Thorold, ON, Canada). The manufacturer’s protocol was followed, with minor modifications [19 ]. All centrifugation steps were increased to 3000× g for 3 min and the final elution (200 µL) was performed following column drying (Step 7). The following kits were also evaluated for purification of total nucleic acids from whole blood: QIAamp DNA Blood Kit (Qiagen, Hilden, Germany), PAXgene Blood RNA System (Qiagen, Hilden, Germany), Preserved Blood RNA Purification Kit (Norgen Biotek, Thorold, ON, Canada), RNAgard Blood System (Biomatrica, San Diego, CA, USA), NucleoSpin RNA Blood Midi (MACHEREY-NAGEL GmbH & Co., Duren, Germany), E.Z.N.A.^® Blood RNA Midi Isolation (Omega Bio-tek, Norcross, GA, USA).