There are several reports on the use of mock clinical specimens when human clinical samples are unavailable. Akyurek et al. prepared SARS-CoV-2 mock clinical specimen by mixing IVT synthesized viral RNA (108 copies/μL) and human total RNA (2.5 ng/μL) in a 1:1 volume ratio53 (link). Park et al. used RVFV mock clinical specimens by mixing RVFV RNA and human tissue RNA54 (link). As we were unable to source human clinical samples of RVFV-infected patients, a mock clinical specimen was prepared to assess the clinical applicability of the developed method. Human adult normal tissue RNA extracted from the blood of a 24-year-old man was obtained from BioChain (Newark, CA, USA). The mock clinical specimen contained 25 μL of human adult normal tissue RNA (200 ng/μL), 25 μL of RVFV IVT RNA, and 200 μL RNA storage solution (Invitrogen). The final concentration of viral genome in this mock clinical specimen was adjusted to 108 copies/mL. The cDNA synthesis, PCR amplification, MinION sequencing, and assembly were performed as described above.
Rna storage solution
Manufactured by Thermo Fisher Scientific
Sourced in United States
RNA Storage Solution is a specialized buffer designed to preserve and protect RNA samples during storage and transportation. It helps maintain the integrity and stability of RNA, preventing degradation and ensuring reliable results in downstream applications.
Automatically generated - may contain errors
Lab products found in correlation
Trizol reagent, by Thermo Fisher Scientific (7 mentions)
Tri reagent, by Molecular Research Center (5 mentions)
Trizol, by Thermo Fisher Scientific (4 mentions)
Qiaamp viral rna mini kit, by Qiagen (4 mentions)
Nanodrop 1000 spectrophotometer, by Thermo Fisher Scientific (4 mentions)
Nanodrop, by Thermo Fisher Scientific (4 mentions)
Rneasy minelute cleanup kit, by Qiagen (3 mentions)
Turbo dnase, by Thermo Fisher Scientific (3 mentions)
Agilent 2100 bioanalyzer, by Agilent Technologies (3 mentions)
Nanodrop 2000, by Thermo Fisher Scientific (2 mentions)
2100 bioanalyzer instrument, by Agilent Technologies (2 mentions)
Megaclear kit, by Thermo Fisher Scientific (2 mentions)
Phenol chloroform extraction, by Merck Group (2 mentions)
Bioanalyzer, by Agilent Technologies (2 mentions)
Taqman gene expression assay probe, by Thermo Fisher Scientific (1 mentions)
Abi 7900ht pcr system, by Thermo Fisher Scientific (1 mentions)
Prep station, by NanoString (1 mentions)
Ncounter, by NanoString (1 mentions)
Superscript 2 reverse transcriptase kit, by Thermo Fisher Scientific (1 mentions)
Direct zol miniprep kit, by Zymo Research (1 mentions)
46 protocols using rna storage solution
1
Preparing RVFV Mock Clinical Specimens
2
Extraction and Purification of Nucleic Acids
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Following pulverization of 40–100 mg of fresh-frozen tissue specimen, total RNA and genomic DNA (gDNA) were extracted using TRI-Reagent (Molecular Research Center, Cincinnati, OH, USA) according to the manufacturer’s instructions. Total RNA was dissolved in RNA Storage Solution (Invitrogen, Carlsbad, CA, USA), and genomic DNA in 8 mM NaOH, pH-adjusted by addition of 0.1 M HEPES buffer. Both RNA and DNA samples were stored at −80°C until analysis. DNA/RNA concentration and purity were determined spectrophotometrically at 260 and 280 nm, while agarose gel electrophoresis was performed to evaluate RNA integrity.
3
Quantitative RNA Expression Analysis
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The total RNA was isolated using the Direct-ZOL Miniprep kit (Zymo Research), and RNA was stored at −80 °C in RNA storage solution (Invitrogen) until subsequent analysis. For real-time PCR, cDNA was synthesized using the SuperScript II Reverse Transcriptase kit (Invitrogen) and anchored oligo-dT primers (Invitrogen). TaqMan Fast Universal-based real-time PCR (Life Technologies) was performed in a ABI 7900HT PCR system (Applied Biosystems), with commercially available TaqMan gene expression assay probes (Thermo Scientific) with GAPDH as an internal normalization control (Supplementary Table 1 ). For NanoString analysis, 50–100 ng of RNA was hybridized to a custom nCounter XT probe set and processed using the NanoString prep station and nCounter (NanoString Technologies). Gene expression levels were determined with NanoString nSolver software with default parameters and normalized with the expression of five housekeeping genes (ITCH, RPL15, RPL19, TCEB1, and UBE2D3). Nanostring data have been deposited into figshare under DOI accession code 10.6084/m9.figshare.7670531.
4
Bacterial RNA Isolation and RT-qPCR
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RNA isolation and RT-qPCR were performed as previously described34 (link). In brief, 5 × 109 bacteria were pelleted and resuspended in 1 ml of TRIzol (Invitrogen) and then lysed using zirconium silica beads. The RNA was then isolated and washed using chloroform, isopropanol and 70% ethanol according to the manufacturer of TRIzol. To solubilize the RNA, the pellet was resuspended in RNA storage solution (Invitrogen), heated at 55 °C for 3-4 min and vortexed. Subsequently, the present DNA was digested by DNase I (Roche). Abscence of DNA contamination was controlled by using QuantiFastSYBR Green-PCR Kit (Qiagen). To quantify mRNA expression the QuantiFastSYBR Green RT-PCR Kit (Qiagen) was used according to the manufacturer´s instructions. RT-qPCR was done using a LightCycler 480 II (Roche). The primers that were used are listed in Supplementary Data 2 .
5
RVFV Lunyo Viral RNA Protocol
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The viral RNA of RVFV Lunyo (EVAg 005N-02174) was obtained from the Department of Health: Public Health England—Virology & Pathogenesis group (DH) through European Virus Archive global (EVAg). The RNA was extracted from the supernatant of a viral-infected cell culture. The RVFV mock clinical sample was prepared by mixing viral RNA and human tissue RNA in an RNA Storage Solution44 (link). The mock samples were prepared with the following composition: 25 µL of RVFV viral RNA, 25 µL (200 ng/µL) of total RNA of Human Adult Normal Tissue: Blood Vessel: Vein (BioChain, USA), and 200 µL of the RNA Storage Solution (Invitrogen, USA).
6
RNA Isolation and RT-PCR Analysis
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For RNA isolation 5 × 109 bacteria were harvested from an overnight culture and resuspended in 1 ml TRIzol Reagent (Thermo Fisher Scientific). RNA isolation and DNase digestion were carried out as described previously (9 (link)). The resulting RNA was stored at a concentration of 0.1 μg/μl in RNA storage solution (Invitrogen) at −80°C until use. To carry out RT-PCR, RNA was thawed on ice and diluted 1:10 using RNase-free water (Ambion). mRNA expression of mutant strains in comparison to wild type was analyzed qualitatively by RT-PCR using the QuantiFast SYBR green qRT-PCR kit (Qiagen) according to the manufacturer’s instructions. The resulting PCR products were analyzed by agarose gel electrophoresis and staining of the gel with SYBR Safe (Thermo Fisher Scientific). Primers used are listed in Table S2.
7
RNA Extraction from Sample Supernatant
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RNA was isolated from 140 μL of sample supernatant using the QIAamp Viral RNA Mini Kit (QIAGEN, Germantown, USA) according to the manufacturer's instructions, except the usage of RNA Storage Solution (Ambion, Foster City, USA) for elution. Extracted RNA was stored at −20°C until use.
8
Total RNA Extraction and mRNA Purification
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Each frozen sample was ground in a mortar with liquid nitrogen, and then total RNA was isolated using TRIzol reagent (Invitrogen, USA) following the manufacture's protocol. The final total RNA was dissolved in 200 µL RNase-free water. The concentration of total RNA was determined using a NanoDrop2000 spectrophotometer (Thermo Scientific, USA), and the RNA integrity was checked using an RNA 6000 Pico LabChip with the Agilent 2100 Bioanalyzer (Agilent, USA). The total RNA was incubated with 10 U DNase I (Ambion, USA) at 37°C for 1 h, and then nuclease-free water was added to dilute the sample volume to 250 µL. The messenger RNA (mRNA) was further purified with a MicroPoly(A) Purist Kit (Ambion, USA) according to the manufacturer's protocol. The mRNA was dissolved in 100 µL of RNA Storage Solution (Ambion). The final concentration was determined using a NanoDrop2000 spectrophotometer.
9
Total RNA Isolation and Quantification
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Total RNA was isolated using TRI-Reagent (Molecular Research Center, Cincinnati, OH, USA) from 40–150 mg of homogenized tissue following the manufacturer’s instructions and, thereafter, was dissolved in RNA Storage Solution (Ambion, Carlsbad, CA, USA). RNA concentration and purity were determined spectrophotometrically at 260 and 280 nm, assessing absorbance ratios at 260/280 nm and 260/230 nm, while the quality of RNA was evaluated by agarose gel electrophoresis.
10
Transcriptomic Analysis of HSV-1 Infection
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Tissue was placed in 500 μl TRIzol reagent (Invitrogen) immediately following removal, snap frozen, and stored in a −80°C freezer. For total RNA extraction, tissue was thawed, homogenized, and processed according to the manufacturer’s instructions. RNA pellets were resuspended in 12 to 30 μl of RNA storage solution (Ambion). Up to 1 μg RNA was converted to cDNA using the iScript Supermix (Bio-Rad) and was processed for RT-PCR using the iTaq Universal SYBR green (Bio-Rad) as previously described [21 (link)]. All genes were normalized to Actb and phosphoglycerate kinase 1 (Pgk1) and quantified by the ∆∆CT method. HSV-1-specific genes, Cxcl10, Ifnγ, and control gene primer sequences have been previously published [21 (link), 22 (link)].
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