Acid-phenol: chloroform: isoamyl alcohol (125:24:1, pH 4.5) or phenol: chloroform: isoamyl alcohol (25:24:1, pH 8.0) was added to an equal volume of the solution intended for RNA or DNA precipitation, respectively. The mixture was vigorously shaken, incubated at room temperature for 5 min, and centrifuged (12,000 g, 15 min, 4 °C). Supernatant was transferred to a new tube and mixed with 0.1 volumes of sodium acetate (3 M, pH 5.2) and 0.01 µl of 20 mg/ml glycogen. The solution was combined with 2.5 volumes of ethanol or an equal volume of isopropanol, and the tube was inverted several times to mix. The sample was kept at −20 °C for at least 1 h until precipitation occurred. After centrifugation (12,000 g, 30 min, 4 °C), the pellet was washed once with 70% ethanol. After drying in air, nuclease-free H2O or THE RNA Storage Solution (Thermo Fisher Scientific, #AM7001) was added to dissolve DNA or RNA into solution.
The rna storage solution
Manufactured by Thermo Fisher Scientific
Sourced in United States
The RNA Storage Solution is a specialized reagent designed to preserve and stabilize RNA samples. It provides an effective means to store RNA samples at reduced temperatures, protecting the integrity of the RNA molecules.
Automatically generated - may contain errors
Lab products found in correlation
Qiaamp viral rna mini kit, by Qiagen (2 mentions)
Spriselect magnetic beads, by Beckman Coulter (2 mentions)
Rna grade glycogen, by Thermo Fisher Scientific (2 mentions)
Taqpath 1 step rt qpcr master mix, by Thermo Fisher Scientific (2 mentions)
Ultrapure guanidine isothiocyanate, by Thermo Fisher Scientific (2 mentions)
Cdc n1 and n2 primer probes 2019 ncov ruo kit, by Integrated DNA Technologies (2 mentions)
Buffer rlt lysis buffer, by Qiagen (2 mentions)
Rnasin plus rnase inhibitor, by Promega (2 mentions)
Proteinase k inhibitor, by Merck Group (2 mentions)
Polyethylene glycol peg bio ultra 8000, by Merck Group (2 mentions)
Proteinase k, by Qiagen (2 mentions)
Trizol reagent, by Thermo Fisher Scientific (2 mentions)
Poly a purist mag kit, by Thermo Fisher Scientific (2 mentions)
Nanodrop one, by Thermo Fisher Scientific (2 mentions)
Rneasy minelute cleanup kit, by Qiagen (1 mentions)
Quantitect reverse transcription kit, by Qiagen (1 mentions)
Superscript 2 reverse transcriptase, by Thermo Fisher Scientific (1 mentions)
Agencourt ampure xp bead, by Beckman Coulter (1 mentions)
Superscript 3 reverse transcriptase, by Thermo Fisher Scientific (1 mentions)
Rnaseout, by Thermo Fisher Scientific (1 mentions)
15 protocols using the rna storage solution
1
RNA/DNA Extraction and Precipitation
2
Annealing and Purification of Small RNA Molecules
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One picomole (pmol) of the BIO and DIG handles and 1.5–2 pmol of the other ssRNAs were used for the annealing reactions, which were set in a total volume of 100 μl, in 8.5 mM sodium citrate and 7.5 mM NaCl, pH 6.4. Annealing was done in a thermocycler as described for ssDNA samples. The double-stranded RNA was purified with the RNeasy MinElute cleanup kit (Qiagen). Hybridization products were eluted with 1 mM sodium citrate (THE RNA Storage Solution, Thermo Fisher Scientific). Concentrations were determined using a Nanodrop.
3
Total RNA Extraction and cDNA Synthesis
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Total RNA was extracted from GC, TL, CL, and positive control samples using TRIzol Reagent according to the manufacturer’s instruction. Pelleted RNA was dissolved in THE RNA Storage
Solution (Thermo Fisher Scientific) at a concentration of 1 µg/µl and kept at −80°C. Removal of contaminating genomic DNA and cDNA synthesis were performed by using the QuantiTect Reverse
Transcription kit (Qiagen, Hilden, Germany) with 2 µg RNA per sample according to the manufacturer’s instruction.
Solution (Thermo Fisher Scientific) at a concentration of 1 µg/µl and kept at −80°C. Removal of contaminating genomic DNA and cDNA synthesis were performed by using the QuantiTect Reverse
Transcription kit (Qiagen, Hilden, Germany) with 2 µg RNA per sample according to the manufacturer’s instruction.
4
SARS-CoV-2 RNA Extraction and Storage
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Purified SARS-CoV-2
RNA used for the development and validation of the multiplex RT-qPCR
assays was provided by our collaborators in the Li Ka Shing Institute
of Virology. The purified RNA was prepared from passaged isolates
of clinical samples submitted to the Provincial Laboratory of Public
Health, Alberta Precision Laboratories (APL) which is an accredited
clinical laboratory responsible for the province-wide clinical testing
of COVID-19. The Global Initiative on Sharing All Influenza Data (GISAID)
accession numbers for the sequences of the SARS-CoV-2 VOCs RNAs extracted
from clinical nasopharyngeal specimens are listed inTable S5 . In brief, the first passage SARS-CoV-2 was made
from 50 μL of an unidentified nasopharyngeal sample, which was
filtered and incubated with Vero-TMPRSS2 cells. The second passage
virus was then made similarly. RNA was extracted using the QIAamp
viral RNA mini kit (Qiagen). The purified RNA was diluted and stored
in THE RNA Storage Solution (Thermo Fisher Scientific, Carlsbad, CA,
USA) containing 1.2 U/μL of RNasin Plus RNase Inhibitor at −80
°C to ensure RNA stability.
RNA used for the development and validation of the multiplex RT-qPCR
assays was provided by our collaborators in the Li Ka Shing Institute
of Virology. The purified RNA was prepared from passaged isolates
of clinical samples submitted to the Provincial Laboratory of Public
Health, Alberta Precision Laboratories (APL) which is an accredited
clinical laboratory responsible for the province-wide clinical testing
of COVID-19. The Global Initiative on Sharing All Influenza Data (GISAID)
accession numbers for the sequences of the SARS-CoV-2 VOCs RNAs extracted
from clinical nasopharyngeal specimens are listed in
from 50 μL of an unidentified nasopharyngeal sample, which was
filtered and incubated with Vero-TMPRSS2 cells. The second passage
virus was then made similarly. RNA was extracted using the QIAamp
viral RNA mini kit (Qiagen). The purified RNA was diluted and stored
in THE RNA Storage Solution (Thermo Fisher Scientific, Carlsbad, CA,
USA) containing 1.2 U/μL of RNasin Plus RNase Inhibitor at −80
°C to ensure RNA stability.
5
SARS-CoV-2 RNA Detection Workflow
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Proteinase K and Buffer RLT lysis buffer were purchased from QIAgen (Germantown, MD, USA). TaqPath 1-Step RT-qPCR Master Mix, CG, UltraPure guanidine isothiocyanate, Gibco Beef Extract Powder, RNA-grade glycogen, and THE RNA Storage Solution were bought from ThermoFisher Scientific (Carlsbad, CA, USA). Polyethylene glycol (PEG) Bio Ultra 8000, glycine (electrophoresis grade), magnesium chloride hexahydrate, and the Proteinase K Inhibitor (tetrapeptidyl chloromethyl ketone) were bought from MilliporeSigma (Oakville, Ontario, Canada). SPRIselect magnetic beads were bought from Beckman Coulter (Brea, CA, USA). 2-Mercaptoethanol (2-ME), biotechnology grade, was bought from BioShop Canada (Burlington, Ontario, Canada). RNasin Plus Rnase Inhibitor was bought from Promega (Madison, WI, USA). Pseudovirus (SARS-CoV-2 RNA targets in a noninfectious viral coat) solution, AccuPlex SARS-CoV-2 Verification Panel-Full Genome, was bought from Sera Care, LGC (Milford, MA, USA). Purified SARS-CoV-2 RNA was provided by our colleagues in the Department of Medical Microbiology and Immunology at the University of Alberta. CDC N1 and N2 primer-probes 2019-nCoV RUO kit was bought from Integrated DNA Technologies (Coralville, IA, USA).
6
SARS-CoV-2 Detection Workflow Using RT-qPCR
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Proteinase K, QIAamp Viral RNA
Mini Kit, and Buffer RLT lysis buffer were purchased from QIAgen (Germantown,
MD, USA). TaqPath 1-Step RT-qPCR Master Mix, CG, UltraPure guanidine
isothiocyanate, RNA-grade glycogen, and THE RNA Storage Solution were
bought from ThermoFisher Scientific (Carlsbad, CA, USA). CDC N1 and
N2 primer-probes 2019-nCoV RUO kit was bought from Integrated DNA
Technologies (Coralville, IA, USA). Proteinase K Inhibitor (tetrapeptidyl
chloromethyl ketone) was bought from MilliporeSigma (Oakville, Ontario,
Canada). RNasin Plus RNase Inhibitor was bought from Promega (Madison,
WI, USA). SPRIselect magnetic beads were bought from Beckman Coulter
(Brea, CA, USA). Silica-coated TurboBeads were ordered from TurboBeadsBio
(Zurich, Switzerland). Polyethylene glycol (PEG) Bio Ultra 8000 was
bought from MilliporeSigma. 2-Mercaptoethanol (2-ME), biotechnology
grade, was bought from BioShop Canada (Burlington, Ontario, Canada).
Pseudo-virus (SARS-CoV-2 RNA targets in a noninfectious viral coat)
solution, AccuPlex SARS-CoV-2 Verification Panel-Full Genome, was
bought from Sera Care, LGC (Milford, MA, USA). QuickExtract RNA Extraction
Kit and QuickExtract Plant DNA Extraction Solution were bought from
Lucigen (Middleton, WI, USA). Purified SARS-CoV-2 RNA was provided
by our colleagues in the Department of Medical Microbiology and Immunology
at the University of Alberta.
Mini Kit, and Buffer RLT lysis buffer were purchased from QIAgen (Germantown,
MD, USA). TaqPath 1-Step RT-qPCR Master Mix, CG, UltraPure guanidine
isothiocyanate, RNA-grade glycogen, and THE RNA Storage Solution were
bought from ThermoFisher Scientific (Carlsbad, CA, USA). CDC N1 and
N2 primer-probes 2019-nCoV RUO kit was bought from Integrated DNA
Technologies (Coralville, IA, USA). Proteinase K Inhibitor (tetrapeptidyl
chloromethyl ketone) was bought from MilliporeSigma (Oakville, Ontario,
Canada). RNasin Plus RNase Inhibitor was bought from Promega (Madison,
WI, USA). SPRIselect magnetic beads were bought from Beckman Coulter
(Brea, CA, USA). Silica-coated TurboBeads were ordered from TurboBeadsBio
(Zurich, Switzerland). Polyethylene glycol (PEG) Bio Ultra 8000 was
bought from MilliporeSigma. 2-Mercaptoethanol (2-ME), biotechnology
grade, was bought from BioShop Canada (Burlington, Ontario, Canada).
Pseudo-virus (SARS-CoV-2 RNA targets in a noninfectious viral coat)
solution, AccuPlex SARS-CoV-2 Verification Panel-Full Genome, was
bought from Sera Care, LGC (Milford, MA, USA). QuickExtract RNA Extraction
Kit and QuickExtract Plant DNA Extraction Solution were bought from
Lucigen (Middleton, WI, USA). Purified SARS-CoV-2 RNA was provided
by our colleagues in the Department of Medical Microbiology and Immunology
at the University of Alberta.
7
Total RNA Extraction and cDNA Synthesis
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Total RNA was extracted from each cell line using the TRIzol Reagent (Ambion™, Thermo Fisher Scientific Inc., Waltham, MA, USA), diluted in THE RNA Storage Solution (Ambion™), and stored at −80 °C until further use. The assessment of purity and concentration of each RNA sample was performed spectrophotometrically at 260 and 280 nm. Then, first-strand cDNA synthesis was performed based on the manufacturer’s guidelines, in reaction volumes of 20 μL, using 5 μg of total RNA from each cell line, SuperScript II Reverse Transcriptase (Invitrogen™, Thermo Fisher Scientific Inc.) and an oligo-dT–adapter as primer (5′-GCGAGCACAGAATTAATACGACTCACTATAGGTTTTTTTTTTTTVN-3′, where V = G, A, C, and N = G, A, T, C).
8
RNA Fragmentation and cDNA Synthesis
9
cDNA Synthesis from Total RNA
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Total RNA was isolated with the use of TRIzol™ Reagent (Ambion™, Thermo Fisher Scientific Inc., Waltham, MA, USA), following the guidelines of the manufacturer. All RNA samples were diluted in THE RNA Storage Solution (Ambion™), whereas their concentration and purity were assessed spectrophotometrically at 260 and 280 nm, using a BioSpec-nano Micro-volume UV-Vis Spectrophotometer (Shimadju, Kyoto, Japan).
In the next step, 2 μg of each RNA sample was used for the implementation of the first-strand cDNA synthesis. For this purpose, an oligo-dT20 was used as RT primer to anneal in the 3’ poly(A) tail of the mRNA molecules. Briefly, the cDNA synthesis was performed in reaction volumes of 20 μL, using 2 μg of total RNA, 1 μL of oligo-dT20 (10 μM), 1 μL dNTP Mix (10 mM each), 4 μL 5X First-Strand Buffer, 1 μL DTT (0.1 M), 1 μL (40 U) RNaseOUT™ (Invitrogen™, Thermo Fisher Scientific Inc., Waltham, MA, USA) and 1 μL (200 U) of SuperScript™ III Reverse Transcriptase (Invitrogen™, Thermo Fisher Scientific Inc., Waltham, MA, USA), according to the manufacturers’ protocol. For the quality control of the produced cDNA samples, the expression levels of the housekeeping gene Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) were tested. Finally, equal amounts of each cDNA were pooled for the creation of a final cDNA mix from the 52 human cell lines.
In the next step, 2 μg of each RNA sample was used for the implementation of the first-strand cDNA synthesis. For this purpose, an oligo-dT20 was used as RT primer to anneal in the 3’ poly(A) tail of the mRNA molecules. Briefly, the cDNA synthesis was performed in reaction volumes of 20 μL, using 2 μg of total RNA, 1 μL of oligo-dT20 (10 μM), 1 μL dNTP Mix (10 mM each), 4 μL 5X First-Strand Buffer, 1 μL DTT (0.1 M), 1 μL (40 U) RNaseOUT™ (Invitrogen™, Thermo Fisher Scientific Inc., Waltham, MA, USA) and 1 μL (200 U) of SuperScript™ III Reverse Transcriptase (Invitrogen™, Thermo Fisher Scientific Inc., Waltham, MA, USA), according to the manufacturers’ protocol. For the quality control of the produced cDNA samples, the expression levels of the housekeeping gene Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) were tested. Finally, equal amounts of each cDNA were pooled for the creation of a final cDNA mix from the 52 human cell lines.
10
Total RNA Extraction from Cells
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Total RNA was extracted from HepG2 cells or primary Dravet fibroblasts using the QIAGEN RNeasy Midi Kit (QIAGEN, USA) as described by the manufacturer. PolyA RNA was isolated with the Poly(A)Purist™ MAG Kit and eluted twice with 200 μl of THE RNA Storage Solution (Ambion, USA).
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