Qubit rna br assay kit

RNA concentration and integrity was measured using the Qubit RNA BR Assay kit (Life Technologies) and an Agilent Technologies 2100 Bioanalyzer, correspondingly. cDNA libraries were constructed with the Illumina TruSeq™ RNA Sample Preparation Kit (Illumina, Inc., San Diego, CA, USA) using 400 ng of total RNA with RNA integrity number (RIN) ≥7. Briefly, the protocol consisted of polyA-RNA enrichment, RNA fragmentation, reverse transcription of fragmented RNA into cDNA, adapters ligation onto both ends of the cDNA fragments and amplification of cDNA fragments by PCR. Resulting cDNA libraries were paired-end sequenced on Illumina HiSeq 4000 by Macrogen (Seoul, Korea) to obtain around 40 million reads per sample.

RNA was purified using RNeasy kits (Qiagen, Hilden, Germany). Total RNA was quantified by a Qubit^® fluorometer using the Qubit^® RNA BR assay kit (Life Technologies, Carlsbad, CA, USA). RNA was reverse-transcribed with the QuantiTect Reverse Transcription Kit (Qiagen, Hilden, Germany) and PCR amplification was performed on a CFX96 Real-time PCR Detection System (BioRad, Hercules, CA, USA) using the QuantiFast SYBR Green PCR Kit (Qiagen, Hilden, Germany). The expression of the genes was normalized to the expression of GAPDH.

C. gigas specimens were sampled in the lagoon of Goro (North Adriatic Sea, Italy) in May 2016. Total flesh of 15 individual oysters (samples S1-S15) was divided in two subsamples, the first one was immediately homogenized in 1 ml Trizol (Life Technologies, Germany) using a T-10 Ultra-Turrax (IKA, Staufen, Germany) and frozen at − 80 °C until RNA extraction, while the second part was subjected to molecular diagnosis of OsHV-1, according to [99 (link)]. The processing of H. diversicolor supertexta samples was described in detail elsewhere, including also the analysis of host and viral expression profiles [68 (link), 78 (link)]. Briefly, the abalones originated from a Chinese farming area and were sampled in the frame of a large AbHV-1 infection trial. The RNA-seq samples originated from time zero, 24 and 60 hpi points of a 0–72 h challenge with an infective homogenate of AbHV-1-CN2003. Total RNA was extracted according to Trizol manufacturer’s instructions, quantified using the Qubit RNA BR Assay Kit (Life Technologies, Carlsbad, USA) and checked for quality using an Agilent Bioanalyzer 2100 Nano kit (Agilent, Santa Clara, USA). cDNA was prepared by retro-transcription of 1 μg of total RNA, using oligo (dT)12–18 primer (25 ng) and 200 U of SuperScript II Reverse Transcriptase (Life Technologies), diluted 1:5 and used for qRT-PCR analysis.

RNA from gill, skin, muscle, stomach, foregut, hindgut, spleen, head kidney, and liver of healthy Atlantic salmon were extracted from frozen (liquid nitrogen and −80 °C) tissue using EZNA total RNA kit (Omega Bio-Tek, Norcross, GA, USA) following the manufacturer’s manual. Extracted RNA was quantified with Qubit^® RNA BR Assay kit and Qubit^® fluorometer (Life technologies, Carlsbad, CA, USA). The total RNA integrity was assessed by running a 1.2% agarose gel. cDNA was synthesized using QuantiTect Reverse Transcription kit with integrated removal of genomic DNA contamination (Qiagen, Germantown, MD, USA) following the manufacturer’s protocol.

RNA was isolated from bacteria using TRIZOL and quantified using Qubit RNA BR Assay Kit (Life technology, Q10210). To quantify over-expressed RNA, 50 ng of total RNA was size separated using RNA ScreenTape Analysis (Agilent, 5067–5576) and quantified using TapeStation software.

The DIG‐labeled RNA probe for each transgene (Gm‐rbcL, Gm‐rbcS, and Gs‐cbbX) was generated from DNA templates with T7 promoters and MEGAshortscript kit (Ambion, Foster City, CA) using the procedure described previously (Occhialini et al., 2016). RNA was extracted from leaf tissues (~ 2‐5 cm²) with PureLink^® RNA Mini Kit (Life Technologies) according to the manufacturer's protocol, and RNA concentrations were estimated with the Qubit^® RNA BR Assay Kit. Denaturing RNA gels with 2% formaldehyde were run with 0.2 μg of each RNA sample, transferred to a supercharged Nylon membrane in 20 x SSC buffer, hybridized with each DIG‐labeled RNA probe at 68°C for 12–16 hr, and detected with alkaline phosphatase‐conjugated anti‐Digoxigenin and CDP‐star chemiluminescent substrate from Roche Life Science (Pleasanton, California) as described previously (Occhialini et al., 2016).

Total RNA was isolated from H. volcanii using the TRIzol method. 50 ml of cells grown in Hv-YPC broth to early log phase (OD₆₅₀ = 0.2) were resuspended in 250 μl of unbuffered spheroplast solution (1 M NaCl, 27 mM KCl, 15% sucrose, pH 7.5) (23 (link)). 750 μl of TRIzol LS (Invitrogen) was added and mixed by pipetting. 200 μl of chloroform (Sigma) was added and vortexed for 15 s, then centrifuged at 20 000 x g for 15 min. The upper phase was transferred to a fresh tube, 500 μl of isopropanol was added and incubated for 10 min at room temperature. RNA was pelleted by centrifugation at 20 000 x g for 10 min, washed with 75% ethanol, air-dried and resuspended in 50 μl of RNase-free dH₂O. Total RNA quality was assessed using the RNA 6000 Nano Kit (Agilent) on an Agilent 2100 Bioanalyzer. Total RNA concentration was measured using Qubit RNA BR assay kit (Life technologies, Q10210). 1 μg of total RNA was used for rRNA depletion using Ribo-Zero rRNA Removal Kit (Illumina). Libraries were prepared using the dUTP method (25 (link)) with NEBNext Ultra Directional RNA Library Prep kit for Illumina (New England Biolabs) and library quality was analyzed on a Bioanalyzer High-Sensitivity DNA-chip (Agilent Biotechnologies). Sequencing was performed on the Illumina NextSeq 500 sequencing platform to generate 2 × 75 bp paired end reads using V2 chemistry.