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TRIpure Reagent(for total RNA isolation)
Cat# GR1021   50ml
Cat# GR1022   100ml
Shipment: Room Temperature
Store at 2 to 8C
TRIpure Reagent has demonstrated stability of 12 months when stored at room temperature. However, we recommend storage at 2 to 8C for optimal performance.
WARNING: This reagent contains pbenol, which will cause burns if it comes in contact with skin. If contact occurs, wash the area with lots of water and detergent.
TRIpure Reagent is a ready-to-use reagent for the isolation of total RNA from samples of mumans,animal, plant, bacterial and viral origin. The TRIpure Reagent is the improved version of the single-step method of RNA isolation. The improved TRIpure Reagent provides a fast and highly reliable method for isolating pure and undegraded RNA from a large variety of biological samples. A biological sample is homogenized or lysed in TRIpure and the homogenate/lysate is separated into aqueous and organic phase by the addition of chloroform. The subsequent centrifugation efficiently removes DNA and proteins from the aqueous phase containing RNA. The undegraded, pure RNA is obtained from the aqueous phase by the isopropanol precipitation, washing with ethanol and solubilization in an appropriate solution. The entire isolation procedure can be completed in 1 hour. The isolated RNA is appropriate for Northern blotting, poly A + selection, RT-PCR, and other molecular biology techniques.
This technique performs well with small quantities of tissue (50-100 mg) and cells (5106), and large quantities of tissue (1 g) and cells (>107), of human, animal, plant, or bacterial origin. The simplicity of the TRIpure Reagent method allows simultaneous processing of a large number of samples.
Precautions for Preventing RNase Contamination:
RNases can be introduced accidentally into the RNA preparation at any point in the isolation procedure through improper technique. Because RNase activity is difficult to inhibit, it is essential to prevent its introduction. The following guidelines should be observed when working with RNA.
• Always wear disposable gloves. Skin often contains bacteria and molds that can contaminate an RNA preparation and be a source of RNases. Practice good microbiological technique to prevent microbial contamination.
• Use sterile, disposable plasticware and automatic pipettes reserved for RNA work to prevent cross-contamination with RNases from shared equipment. For example, a laboratory that is using RNA probes will likely be using RNase A or T1 to reduce background on filters, and any nondisposable items (such as automatic pipettes) can be rich sources of RNases.
• In the presence of TRIpure Reagent, RNA is protected from RNase contamination. Downstream sample handling requires that nondisposable glassware or plasticware be RNase-free. Glass items can be baked at 150C for 4 hours, and plastic items can be soaked for 10 minutes in 0.5 M NaOH, rinsed thoroughly with water, and autoclaved.
Other Precautions:
• Use of disposable tubes made of clear polypropylene is recommended when working with less than 2-ml volumes of TRIpure Reagent.
• For larger volumes, use glass (Corex) or polypropylene tubes, and test to be sure that the tubes can withstand 12,000g with TRIpure Reagent and chloroform. Do not use tubes that leak or crack.
• Carefully equilibrate the weights of the tubes prior to centrifugation.
• Glass tubes must be sealed with parafilm topped with a layer of foil, and polypropylene tubes must be capped before centrifugation.
Caution: When working with TRIpure Reagent use gloves and eye protection (shield, safety goggles). Avoid contact with skin or clothing. Use in a chemical fume hood. Avoid breathing vapor. Unless otherwise stated, the procedure is carried out at 15 to 30C, and reagents are at 15 to 30C.
Reagents required, but not supplied:
• Chloroform
• Isopropyl alcohol
• 75% Ethanol (in DEPC-treated water)
• RNase-free water or 0.5% SDS solution [To prepare RNase-free water, draw water into
RNase-free glass bottles. Add diethylpyrocarbonate (DEPC) to 0.01% (v/v). Let stand overnight and autoclave. The SDS solution must be prepared using DEPC-treated, autoclaved water.
a. Tissues
Homogenize tissue samples in 1 ml of TRIpure Reagent per 50-100 mg of tissue using a glass-Teflon or power homogenizer. The sample volume should not exceed 10% of the volume of TRIpure Reagent used for homogenization. Or transfer 50-100mg of tissue to a mortar and homogenize in liquid nitrogen and add 1 ml of TRIpure Reagent, mix well the homogenized.
b. Cells Grown in Monolayer
Lyse cells directly in a culture dish by adding 1 ml of TRIpure Reagent to a 3.5 cm diameter dish, and passing the cell lysate several times through a pipette. The amount of TRIpure Reagent added is based on the area of the culture dish (1ml per 10cm2) and not on the number of cells present. An insufficient amount of TRIpure Reagent may result in contamination of the isolated RNA with DNA.
c. Cells Grown in Suspension
Pellet cells by centrifugation. Lyse cells in TRIpure Reagent by repetitive pipetting. Use
1 ml of the reagent per 5-10106 of animal, plant or yeast cells, or per 1107 bacterial cells. Washing cells before addition of TRIpure Reagent should be avoided as this increases the possibility of mRNA degradation. Disruption of some yeast and bacterial cells may require the use of a homogenizer.
OPTIONAL: An additional isolation step may be required for samples with high content of proteins, fat, polysaccharides or extracellular material such as muscles, fat tissue, and tuberous parts of plants. Following homogenization, remove insoluble material from the homogenate by centrifugation at 12,000g for 10 minutes at 2 to 8C. The resulting pellet contains extracellular membranes, polysaccharides, and high molecular weight DNA, while the supernatant contains RNA. In samples from fat tissue, an excess of fat collects as a top layer which should be removed. In each case, transfer the cleared homogenate solution to a fresh tube and proceed with chloroform addition and phase separation as described.
Incubate the homogenized samples for 5 minutes at 15 to 30C to permit the complete dissociation of nucleoprotein complexes. Add 0.2 ml of chloroform per 1 ml of TRIpure Reagent. Cap sample tubes securely. Shake tubes vigorously by hand for 15 seconds and incubate them at 15 to 30C for 2 to 3 minutes. Centrifuge the samples at no more than 12,000g for 15 minutes at 2 to 8C. Following centrifugation, the mixture separates into a lower red, phenol-chloroform phase, an interphase, and a colorless upper aqueous phase. RNA remains exclusively in the aqueous phase. The volume of the aqueous phase is about 60% of the volume of TRIpure Reagent used for homogenization.
Transfer the aqueous phase to a fresh tube, and save the organic phase if isolation of DNA or protein is desired. Precipitate the RNA from the aqueous phase by mixing with isopropyl alcohol. Use 0.5 ml of isopropyl alcohol per 1 ml of TRIpure Reagent used for the initial homogenization. Incubate samples at 15 to 30C for 10 minutes and centrifuge at no more than 12,000g for 10 minutes at 2 to 8C. The RNA precipitate, often invisible before centrifugation, forms a gel-like pellet on the side and bottom of the tube.
Remove the supernatant. Wash the RNA pellet once with 75% ethanol, adding at least 1 ml of 75% ethanol per 1 ml of TRIpure Reagent used for the initial homogenization. Mix the sample by vortexing and centrifuge at no more than 12,000g for 5 minutes at 2 to 8C.
At the end of the procedure, briefly dry the RNA pellet (air-dry or vacuum-dry for 5-10
minutes). Do not dry the RNA by centrifugation under vacuum. It is important not to let the RNA pellet dry completely as this will greatly decrease its solubility. Partially dissolved RNA samples have an A260/280 ratio < 1.6. Dissolve RNA in RNase-free water or 0.5% SDS solution by passing the solution a few times through a pipette tip, and incubating for 10 minutes at 55 to 60C. (Avoid SDS when RNA will be used in subsequent enzymatic reactions.) RNA can also be redissolved in 100% formamide (deionized) and stored at -70C.
RNA Isolation Notes:
1. Isolation of RNA from small quantities of tissue (1 to 10 mg) or Cell (102 to 104) Samples: Add 800µl of TRIpure Reagent to the tissue or cells. Following sample lysis, add chloroform and proceed with the phase separation as described in step 2. Prior to precipitating the RNA with isopropyl alcohol, add 5-10 µg RNase-free glycoge as carrier to the aqueous phase. To reduce viscosity, shear the genomic DNA with 2 passes through a 26 gauge needle prior to chloroform addition. The glycogen remains in the aqueous phase and is co-precipitated with the RNA. It does not inhibit first-strand synthesis at concentrations up to 4 mg/ml and does not inhibit PCR.
2. After homogenization and before addition of chloroform, samples can be stored at -60 to -70C for at least one month. The RNA precipitate (step 4, RNA WASH) can be stored in 75% ethanol at 2 to 8C for at least one week, or at least one year at C5 to -20C.
3. Table-top centrifuges that can attain a maximum of 2,600g are suitable for use in these protocols if the centrifugation time is increased to 30-60 minutes in steps 2 and 3.
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