Viral/total RNA was extracted from plasma/sera and tissues, using Qiamp viral RNA (Qiagen) and RNeasy lipid tissue RNA extraction kit (Qiagen), respectively. Protocol as outlined by the kit was followed, except for tissue harvested from CTR rabbits. For these samples, Qiazol (Qiagen) was substituted with Trizol (Invitrogen). Tissue was homogenized in 1 mL of Qiazol or Trizol using an Omni-tip homogenizer (Omni) or stainless steel beads with a mixer mill (Retsch, Inc., Newtown, PA, USA), respectively. The remaining steps were performed according to the manufacturer’s protocol. Additional DNase I digest in solution was performed on extracted RNA, followed by additional RNA clean up with RNeasy RNA extraction kit (Qiagen). The absence of genomic DNA was confirmed by performing PCR without reverse-transcription on a selection of RNA samples from each extraction batch. Quality and concentration of extracted RNA was determined by Nanodrop 1000 spectrophotometer (Thermo Scientific). RNA samples were kept at −80 °C.
Trizol
Manufactured by Omni International
Sourced in United States
Trizol is a reagent used for the isolation and purification of RNA from biological samples. It is a monophasic solution of phenol, guanidine isothiocyanate, and other proprietary components that facilitates the extraction and separation of RNA, DNA, and proteins from cells and tissues.
Automatically generated - may contain errors
Lab products found in correlation
Trizol, by Thermo Fisher Scientific (6 mentions)
Rneasy mini kit, by Qiagen (2 mentions)
Nanodrop 1000 spectrophotometer, by Thermo Fisher Scientific (2 mentions)
Rneasy rna extraction kit, by Qiagen (1 mentions)
Qiamp viral rna, by Qiagen (1 mentions)
Rneasy lipid tissue rna extraction kit, by Qiagen (1 mentions)
Omni tip homogenizer, by Omni International (1 mentions)
Qiazol, by Omni International (1 mentions)
Qiazol, by Qiagen (1 mentions)
Nanodrop 8000 spectrophotometer, by Thermo Fisher Scientific (1 mentions)
Omni prep, by Omni International (1 mentions)
Bioanalyzer, by Agilent Technologies (1 mentions)
Rna 6000 nano kit, by Agilent Technologies (1 mentions)
Glycoblue, by Thermo Fisher Scientific (1 mentions)
Omni tissue homogenizer, by Omni International (1 mentions)
Agilent rna 600 nano kit, by Agilent Technologies (1 mentions)
Agilent 2100 bioanalyzer, by Agilent Technologies (1 mentions)
7 mm probe, by Omni International (1 mentions)
Tissue master 125 lab homogenizer, by Omni International (1 mentions)
Centrifuge 5417r, by Eppendorf (1 mentions)
5 protocols using trizol
1
Optimized RNA Extraction from Plasma/Tissue
2
Efficient Hepatic RNA Extraction Protocol
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Hepatic RNA was isolated according to the manufacturer’s protocol for Trizol (Ambion, Carlsbad, CA, USA), with a few exceptions. Briefly, a six-station Omni Prep homogenizer (Omni International, Kennesaw, GA, USA) was used to homogenize 50–100 mg of liver in 1 mL of Trizol for 40 s. The first centrifugation was lengthened to 20 min (at 11,750 rcf) from the standard 15 min, and the pelleted RNA was washed twice with 70% ethanol. The RNA pellets were suspended in 50–150 µL of nuclease-free water, depending on the size of the pellet. Genomic DNA was removed from the extracted RNA using the Qiagen RNeasy mini-kit (Valenci, CA, USA) with a gDNA eliminator column, according to the manufacturer’s protocol. A Nanodrop 8000 spectrophotometer (Thermo Scientific, Wilmington, DE, USA) was used to measure the concentration of the total RNA. The average 260/280 ratio was 1.93 (range 1.91–2.15). The RNA integrity number was measured using an Agilent Bioanalyzer and a RNA 6000 Nano kit (Santa Clara, CA, USA). The range of the RNA integrity numbers (RIN) was 4.9–8.7, with an average of 6.7. All samples had a RIN of 6.0 or higher, except two samples, which had RINs of 4.9 and 5.9.
3
Total RNA Extraction from Liver Tissue
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Total RNA was extracted using TRIzol® (Life Technologies) as per the manufacturer’s instructions. Briefly, 2-3 mg of frozen liver tissue was pulverized under liquid nitrogen and 800 μl of TRIzol® was added to the resultant powder which was then homogenised with an Omni Tissue Homogenizer (Omni International). 200 μl of chloroform was added to separate the mixture into a red phenol-chloroform phase, an interphase and an RNA containing colourless upper aqueous phase. The aqueous phase was collected and total RNA was precipitated with isopropyl alcohol and GlycoBlue (50 μg/mL) (Life Technologies). The resultant RNA pellet was washed with 75% ethanol, air dried and re-suspended in 20 μl DEPC-treated water. RNA was stored at −80°C prior to quality control and use in first strand cDNA synthesis. Quality and quantity of RNA was analyzed on the 2100 Agilent Bioanalyzer, using the Agilent RNA 600 Nano kit and BioSizing software (Agilent Technologies).
4
RNA Extraction from Tissue Samples
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At the appropriate time point, the colon was removed from euthanized animals and placed directly in 6 ml of TRIzol (Thermo Fisher Scientific, 15596026) in a 50 ml falcon tube. Samples were homogenized in TRIzol using the Tissue Master 125 Lab Homogenizer with a 7 mm probe (Omni International, TM125-115). One milliliter of the TRIzol homogenate was placed into a 1.5 ml microfuge tube containing 200 μl of chloroform. Samples were vigorously shaken, incubated at room temperature for 2–3 min, and centrifuged at 12,000 rcf for 10 min. Five hundred microliters of the aqueous (top) phase was placed into a 2.0 ml microfuge tube that contained 500 μl isopropanol and 20 ng/μl glycogen, and mixed by inversion. Samples were stored as a mixture with isopropanol at −20 °C until further processing. The RNA samples in isopropanol were precipitated and washed with 75% ethanol following the TRIzol Reagent User Guide. Samples were resuspended in 100 μl of RNase-free water and stored in 10 μl aliquots at −80 °C.
5
RNA Isolation from Tooth Pulp Tissue
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RNA was isolated from maxillary incisor tooth pulp tissue of all animals and conserved by immediate freezing in liquid nitrogen. The frozen pulp was homogenized separately in 1.5mL
PCR grade tube containing 1ml TRIZOL (life technology, Zug, Switzerland) at 4°C using a homogenizer (Omni international, Kennesaw, USA) and were left to solubilize in the TRIZOL for 5 min. Subsequently, 200 µL of chloroform (Sigma-Aldrich, Buchs, Switzerland) was added and the specimens were shaken vigorously for 15 sc followed by 5 min incubation at room temperature. The tubes were then centrifuged for 15 min, 14800 gat 4°C (centrifuge 5417R, Eppendorf, Hambourg, Germany) to yield a three layered solution. The RNA phase was transferred to a new tube and precipitated with an equal volume of isopropanol for 10 min at room temperature. Centrifugation (14800 g at 4°C) for 15 min was again performed to collect the RNA pellet. Supernatant was discarded and 1mL of 75% (v/v) ethanol was added to wash the pellet followed by centrifugation (7500 g for 5 min).
The ethanol was discarded before RNA purification with RNeasy mini kit (Qiagen, Hilden, Germany). The quantity and quality of the RNA was checked before storage at -20°C by Nano drop and 1% agarose gel.
PCR grade tube containing 1ml TRIZOL (life technology, Zug, Switzerland) at 4°C using a homogenizer (Omni international, Kennesaw, USA) and were left to solubilize in the TRIZOL for 5 min. Subsequently, 200 µL of chloroform (Sigma-Aldrich, Buchs, Switzerland) was added and the specimens were shaken vigorously for 15 sc followed by 5 min incubation at room temperature. The tubes were then centrifuged for 15 min, 14800 gat 4°C (centrifuge 5417R, Eppendorf, Hambourg, Germany) to yield a three layered solution. The RNA phase was transferred to a new tube and precipitated with an equal volume of isopropanol for 10 min at room temperature. Centrifugation (14800 g at 4°C) for 15 min was again performed to collect the RNA pellet. Supernatant was discarded and 1mL of 75% (v/v) ethanol was added to wash the pellet followed by centrifugation (7500 g for 5 min).
The ethanol was discarded before RNA purification with RNeasy mini kit (Qiagen, Hilden, Germany). The quantity and quality of the RNA was checked before storage at -20°C by Nano drop and 1% agarose gel.
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