TRIzol Reagent

Catalog Numbers
15596-026 15596-018

Quantity
100 mL 200 mL

Store at room temperature

Description
TRIzol Reagent is a ready-to-use reagent, designed to isolate high quality total RNA (as well as DNA and proteins) from cell and tissue samples of human, animal, plant, yeast, or bacterial origin, within one hour. TRIzol Reagent is a monophasic solution of phenol, guanidine isothiocyanate, and other proprietary components which facilitate the isolation of a variety of RNA species of large or small molecular size. TRIzol Reagent maintains the integrity of the RNA due to highly effective inhibition of RNase activity while disrupting cells and dissolving cell components during sample homogenization. TRIzol Reagent allows for simultaneous processing of a large number of samples, and is an improvement to the single-step RNA isolation method developed by Chomcynski and Sacchi (Chomczynski & Sacchi, 1987). TRIzol Reagent allows the user to perform sequential precipitation of RNA, DNA, and proteins from a single sample (Chomczynski, 1993). After homogenizing the sample with TRIzol Reagent, chloroform is added, and the homogenate is allowed to separate into a clear upper aqueous layer (containing RNA), an interphase, and a red lower organic layer (containing the DNA and proteins). RNA is precipitated from the aqueous layer with isopropanol. DNA is precipitated from the interphase/organic layer with ethanol. Protein is precipitated from the phenol-ethanol supernatant by isopropanol precipitation. The precipitated RNA, DNA, or protein is washed to remove impurities, and then resuspended for use in downstream applications. Isolated RNA can be used in RT-PCR, Northern Blot analysis, Dot Blot hybridization, poly(A)+ selection, in vitro translation, RNase protection assay, and molecular cloning. Isolated DNA can be used in PCR, Restriction Enzyme digestion, and Southern Blots. Isolated protein can be used for Western Blots, recovery of some enzymatic activity, and some immunoprecipitation.

Caution
TRIzol Reagent contains phenol (toxic and corrosive) and guanidine isothiocyanate (an irritant), and may be a health hazard if not handled properly. Always work with TRIzol Reagent in a fume hood, and always wear a lab coat, gloves and safety glasses. Contact your Environmental Heath and Safety (EH&S) department for proper work and disposal guidelines. Avoid direct contact with TRIzol Reagent, because contact to skin, eyes, or respiratory tract may cause chemical burns to the exposed area. If contact to skin or eyes occurs, immediately wash the exposed area with copious amounts of water for 15 minutes and seek medical attention if necessary. If you inhale vapors, move to fresh air and seek medical attention if necessary. For more information, refer to the TRIzol Reagent SDS (Safety Data Sheet), available from our web site at www.invitrogen.com/sds.

Contents and Storage

TRIzol Reagent is supplied in 100 mL (Cat. no. 15596-026) or 200 mL (Cat. no. 15596-018) volumes, and shipped at room temperature. Upon receipt, store TRIzol Reagent at room temperature. TRIzol Reagent is stable for 12 months when properly stored.

Intended Use
For research use only. Not intended for human or animal diagnostic or therapeutic uses.

Materials Needed
The following additional materials are needed, but not supplied for the isolation of RNA, DNA or proteins.
RNA Isolation Chloroform Isopropyl alcohol 75% ethanol (in DEPC-treated water) RNase-free water or 0.5% SDS Centrifuge and rotor capable of reaching up to 12,000 g Polypropylene microcentrifuge tubes Water bath or heat block (5560C) DNA Isolation Chloroform 100% ethanol 75% ethanol 0.1 M sodium citrate in 10% ethanol 8 mM NaOH Centrifuge and rotor capable of reaching up to 12,000 g Polypropylene microcentrifuge tubes Protein Isolation Chloroform Isopropyl alcohol 100% ethanol 0.3 M Guanidine hydrochloride in 95% ethanol 1% SDS Centrifuge and rotor capable of reaching up to 12,000 g Polypropylene microcentrifuge tubes

Part no. 15596026.PPS

MAN0001271

Rev. Date: Date: 15 Nov 2010

For support, visit www.invitrogen.com/support or email [email protected]. To reorder, visit www.invitrogen.com.

Preparing Samples
Homogenizing samples
1. Determine your sample type, and perform homogenization at room temperature according to the table below. The sample volume should not exceed 10% of the volume of TRIzol Reagent used for homogenization. Be sure to use the indicated amount of TRIzol Reagent, because an insufficient volume can result in DNA contamination of isolated RNA.
Sample Type Tissues Action 1. 2. Add 1 mL TRIzol Reagent per 50100 mg of tissue sample. Homogenize sample using a glassTeflon or power homogenizer. Note: Process or freeze tissue samples immediately upon collection. Remove growth media from culture dish. Add 1 mL TRIzol Reagent directly to the cells in the culture dish per 10 cm2 of culture dish surface area. Note: Add 1 mL TRIzol Reagent for a 35 mm dish, 3 mL for a 60 mm dish, and 8 mL for a 100 mm dish. Lyse the cells directly in the culture dish by pipetting the cells up and down several times. Harvest cells by centrifugation and remove media. Add 0.75 mL of TRIzol Reagent per 0.25 mL of sample (510 106 cells from animal, plant or yeast origin, or 1 107 cells of bacterial origin). Note: Do not wash cells before addition of TRIzol Reagent to avoid increased chance of mRNA degradation. Lyse cells in sample by pipetting up and down several times. Disruption of some yeast and bacterial cells may require the use of a homogenizer.

Phase separation
1. Incubate the homogenized sample (see Homogenizing samples) for 5 minutes at room temperature to permit complete dissociation of the nucleoprotein complex. Add 0.2 mL of chloroform per 1 mL of TRIzol Reagent used for homogenization. Cap the tube securely. Shake tube vigorously by hand for 15 seconds. Incubate for 23 minutes at room temperature. Centrifuge the sample at 12,000 g for 15 minutes at 4C. Note: The mixture separates into a lower red phenolchloroform phase, an interphase, and a colorless upper aqueous phase. RNA remains exclusively in the aqueous phase. The upper aqueous phase is ~50% of the total volume. Remove the aqueous phase of the sample by angling the tube at 45 and pipetting the solution out. Avoid drawing any of the interphase or organic layer into the pipette when removing the aqueous phase. Place the aqueous phase into a new tube and proceed to the RNA Isolation Procedure. Save the interphase and organic phenol-chloroform phase if isolation of DNA or protein is desired. See DNA Isolation Procedure and Protein Isolation Procedure for details. The organic phase can be stored at 4C overnight.

2. 3. 4. 5.

6.

Adherent Cells (Monolayer)

1. 2.

7. 8.

3.

Suspension Cells

1. 2.

RNA Isolation Procedure

Always use the appropriate precautions to avoid RNase contamination when preparing and handling RNA.

RNA precipitation
1. (Optional) When precipitating RNA from small sample quantities (<106 cells or <10 mg tissue), add 510 g of RNase-free glycogen as a carrier to the aqueous phase. Note: Glycogen is co-precipitated with the RNA, but does not inhibit first-strand synthesis at concentrations 4 mg/mL, and does not inhibit PCR. Add 0.5 mL of 100% isopropanol to the aqueous phase, per 1 mL of TRIzol Reagent used for homogenization. Incubate at room temperature for 10 minutes. Centrifuge at 12,000 g for 10 minutes at 4C. Note: The RNA is often invisible prior to centrifugation, and forms a gel-like pellet on the side and bottom of the tube. Proceed to RNA wash. Remove the supernatant from the tube, leaving only the RNA pellet. Wash the pellet, with 1 mL of 75% ethanol per 1 mL of TRIzol Reagent used in the initial homogenization. Note: The RNA can be stored in 75% ethanol at least 1 year at 20C, or at least 1 week at 4C. Vortex the sample briefly, then centrifuge the tube at 7500 g for 5 minutes at 4C. Discard the wash. Vacuum or air dry the RNA pellet for 510 minutes. Do not dry the pellet by vacuum centrifuge. Note: Do not allow the RNA to dry completely, because the pellet can lose solubility. Partially dissolved RNA samples have an A260/280 ratio <1.6. Proceed to RNA resuspension.

3.

2. 3. 4.

2.

(Optional) When preparing samples with high content of fat, proteins, polysaccharides, or extracellular material (e.g., muscle, fat tissue, or tuberous plant material), an additional isolation step may be required to remove insoluble material from the samples. Note: Do not perform this additional isolation step if you are performing subsequent DNA isolation on your sample.
Sample Type Tissue or cells with high content of fat, protein, polysaccharide, or extracellular material Notes 1. Following homogenization, centrifuge your sample at 12,000 g for 10 minutes at 4C. Note: The resulting pellet contains ECM, polysaccharides, and high molecular weight DNA, while the supernatant contains the RNA. In high fat content samples, a layer of fat collects above the supernatant. 2. Remove and discard the fatty layer. 3. Transfer the cleared supernatant to a new tube.

5. 1. 2.

RNA wash

3.
4.

3.

Proceed to Phase separation, or store the homogenized sample. Homogenized samples can be stored at room temperature for several hours, or at 60 to 70C for at least one month.

5.

RNA resuspension
1. Resuspend the RNA pellet in RNase-free water or 0.5% SDS solution (2050 L) by passing the solution up and down several times through a pipette tip. Note: Do not dissolve the RNA in 0.5% SDS if it is to be used in subsequent enzymatic reactions. Incubate in a water bath or heat block set at 5560C for 1015 minutes. Proceed to downstream application, or store at 70C.

DNA resuspension
Resuspend the DNA in 8mM NaOH at a concentration of 0.20.3 g/L. 1. Add 0.30.6 mL of 8mM NaOH per 5070 mg of tissue, or per 1 107 cells. Note: Resuspending the DNA is a mild base is highly recommended because isolated DNA does not resuspend well in water or Tris buffer. 2. Remove any insoluble material by centrifuging the sample at 12,000 g for 10 minutes at 4C. 3. Transfer the supernatant containing the DNA to a new tube. Adjust pH as needed with HEPES and proceed to downstream application of choice. The DNA can be stored overnight at 4C, but for long-term storage adjust to pH 78 with HEPES, and add 1 mM EDTA. Store at 4C or 20C.

2. 3.

DNA Isolation Procedure

DNA is isolated from the interphase and phenol-chloroform layer saved from the Phase separation step.

DNA precipitation
1. Remove any remaining aqueous phase overlying the interphase. This is critical for the quality of the isolated DNA. Add 0.3 mL of 100% ethanol per of 1 mL TRIzol Reagent used for the initial homogenization. Cap the tube and invert the sample several times to mix. Incubate samples for 23 minutes at room temperature. Centrifuge the tube at 2000 g for 5 minutes at 4C to pellet the DNA. Remove the phenol-ethanol supernatant and save it in a new tube if protein isolation is desired. The supernatant can be stored at 70C for several months. Proceed with the DNA wash step using the DNA pellet. Wash the DNA pellet with 1 mL of sodium citrate/ ethanol solution (0.1 M sodium citrate in 10% ethanol, pH 8.5) per 1 mL of TRIzol Reagent used for the initial homogenization. Incubate for 30 minutes at room temperature. Mix occasionally by gentle inversion. Note: The DNA can be stored in sodium citrate/ethanol solution at least 2 hours. Centrifuge at 2000 g for 5 minutes at 4C. Remove and discard supernatant. Repeat wash (steps 13), once. Note: Repeat wash twice for large DNA pellets (>200 g). Add 1.52 mL 75% ethanol per 1 mL of TRIzol Reagent used for the initial homogenization. Note: DNA samples may be stored in 75% ethanol at 4C for several months. Incubate for 1020 minutes at room temperature. Mix the tube occasionally by gentle inversion. Centrifuge at 2000 g for 5 minutes at 4C. Remove and discard supernatant. Air or vacuum dry the DNA pellet for 510 minutes. Do not allow the pellet to dry out. Do not dry the pellet by vacuum centrifuge. Proceed to the DNA resuspension step.

Determining Yield of RNA and DNA

Use absorbance of RNA and DNA at 260 nm and 280 nm to determine concentration.
Sample RNA Procedure 1. Dilute sample in RNase-free water, and measure absorbance at 260 nm and 280 nm. 2. Use the formula A260 dilution 40 = g RNA/mL to determine concentration. 1. Dilute sample in water or buffer (pH >7.5), and measure absorbance at 260 nm and 280 nm. 2. Use the formula A260 dilution 50 = g DNA/mL to determine concentration.

2. 3. 4. 5. 6.

DNA

7. 1.

DNA wash

Expected yields
The table below presents typical yields of RNA (A260/280 of >1.8) and DNA (A260/280 of 1.61.8) from various starting materials.
Starting Material Epithelial Cells New Tobacco Leaf Fibroblasts Cultured cells, mammal Skeletal muscles and brain Placenta Liver Kidney Quantity 1 106 cells 1 106 cells 1 106 1 mg 1 mg 1 mg 11.5 g 14 g 610 g 34 g RNA 815 g 73 g 57 g DNA 57 g 57 g 23 g 23 g 34 g 34 g

2.

3. 4. 5.

Protein Isolation Procedure

Proteins are isolated from the phenol-ethanol supernatant layer left over after the DNA precipitation step. Isolate the protein using either Protein precipitation OR Protein dialysis.

6. 7. 8.

Protein precipitation
1. Add 1.5 mL of isopropanol to the phenol-ethanol supernatant per of 1 mL TRIzol Reagent used for the initial homogenization. Incubate samples for 10 minutes at room temperature. Centrifuge at 12,000 g for 10 minutes at 4C to pellet the protein. Remove and discard the supernatant.

2. 3.

9.

4.

Proceed to the Protein wash step with the remaining protein pellet.

Protein wash
Prepare a wash solution consisting of 0.3 M guanidine hydrochloride in 95% ethanol. 2. Wash the protein pellet with 2 mL of the wash solution per 1 mL of TRIzol Reagent used for the initial homogenization. 3. Incubate for 20 minutes at room temperature. Note: Protein samples may be stored in 0.3 M guanidine hydrochloride-95% ethanol for at least one month at 4C or for at least one year at 20C. 4. Centrifuge at 7500 g for 5 minutes at 4C. Remove and discard the wash solution. 5. Repeat steps 24, two more times. 6. Add 2 mL of 100% ethanol to protein pellet after the third wash and vortex. 7. Incubate for 20 minutes at room temperature. 8. Centrifuge at 7500 g for 5 minutes at 4C. Remove and discard ethanol wash. 9. Air dry the protein pellet for 510 minutes. Do not allow the pellet to dry out. 10. Proceed to the Protein resuspension step. 1.

Protein resuspension, continued

Poor solubility of the pellet in SDS can occur, because the solubility of specific classes of proteins differs with different solvents. If the protein pellet is insoluble in SDS, the following alternative solvents (Hummon et. al., 2007) may be required to solubilize the pellet: 1. 1% SDS and 62.5 mM sarkosyl at pH 8.08.8 9.5 M urea and 2% CHAPS, pH 9.1 250mM glycerol, 10mM TEA, and 4% CHAPS 2% diethylamine 10M Urea Load the phenol-ethanol supernatant into the dialysis membrane. Note: The phenol-ethanol solution can dissolve some types of dialysis membranes (e.g., cellulose ester). Test dialysis tubing with the membrane to assess compatibility before starting. Dialyze the sample against 3 changes of 0.1% SDS at 4C. Make the first change of solution after 16 hours, the second change 4 hours later (at 20 hours), and the final change 2 hours later (at 22 hours). Note: 0.1% SDS is required to resolubilize the proteins from the pellet; a lower concentration of SDS is insufficient. If desired, the SDS can be diluted after solubilization. Centrifuge the dialysate at 10,000 g for 10 minutes at 4C. Proteins are located in the clear supernatant. Transfer supernatant to a new tube and proceed to downstream application, or store the sample at 20C. (Optional) Solubilize the pellet by adding 100 L of 1% SDS and 100 L of 8 M urea.

Protein dialysis

2.

Protein resuspension
1.
Add 1% SDS to the protein pellet (200 L) and pipet up and down until the protein is resuspend. Note: To completely dissolve the protein pellet, you may need to incubate the sample at 50C in a water bath or heat block. Centrifuge at 10,000 g for 10 minutes at 4C to sediment any insoluble material. Transfer the supernatant containing the protein to a new tube and proceed to downstream application of choice, or store the sample at 20C. 3. 4. 5.

2. 3.

Determining Yield of Protein

Measure protein concentration by Bradford assay (SDS concentration must be <0.1%).

Troubleshooting
Problem Low yield of RNA, Low yield of DNA, or Low yield of protein Cause Incomplete homogenization or lysis of samples Final RNA, DNA or protein pellet was incompletely redissolved RNA is degraded, DNA is degraded, or Protein is degraded. RNA contamination or DNA contamination Samples were not immediately processed or frozen after collection. Isolated RNA, DNA, or protein preparations were stored at the incorrect temperature. Interphase/organic phase pipetted up with aqueous phase. Incomplete removal of aqueous phase DNA pellet insufficiently washed with 0.1 M sodium citrate in 10% ethanol Low A260/280 for RNA Sample was homogenized in an insufficient volume of TRIzol Reagent Incomplete removal of organic phase Phenol was not sufficiently removed from the DNA preparation Solution Decrease the amount of starting material. Mince tissues into smaller pieces and make sure it is completely immersed in TRIzol Reagent for optimal lysis. Increase the solubilization rate by pipetting the sample repeatedly, and heat the sample to 5060C. Sample must be processed or frozen immediately after collection. Store RNA samples at 60 to 70C. Store DNA and protein samples at 20C. Do not attempt to draw off the entire aqueous layer after phase separation. Remove remnants of the aqueous phase prior to DNA precipitation. Make sure pellet is washed with 0.1 M sodium citrate in 10% ethanol. Add the appropriate amount of TRIzol Reagent for your sample type (see Homogenizing samples). Do not attempt to draw off the entire aqueous layer after phase separation. Wash the DNA pellet one additional time in 0.1 M sodium citrate in 10% ethanol.

Low A260/280 for DNA

References:

Chomczynski, P. (1993) A reagent for the single-step simultaneous isolation of RNA, DNA and proteins from cell and tissue samples. BioTechniques 15, 532-537 Chomczynski, P., and Sacchi, N. (1987) Single Step Method of RNA Isolation by Acid Guanidinium Thiocyanate-Phenol-Chloroform Extraction. Anal. Biochem. 162, 156-159 Hummon, A. B., Lim S. R., Difilippantonio, M. J., Ried, T. (2007) Isolation and solubilization of proteins after TRIzol extraction of RNA and DNA from patient material following prolonged storage. BioTechniques 42, 467-472
Limited Use Label License No. 358: Research Use Only: The purchase of this product conveys to the purchaser the limited, non-transferable right to use the purchased amount of the product only to perform internal research for the sole benefit of the purchaser. No right to resell this product or any of its components is conveyed expressly, by implication, or by estoppel. This product is for internal research purposes only and is not for use in commercial services of any kind, including, without limitation, reporting the results of purchasers activities for a fee or other form of consideration. For information on obtaining additional rights, please contact [email protected] or Out Licensing, Life Technologies, 5791 Van Allen Way, Carlsbad, California 92008. 2010 Life Technologies Corporation. All rights reserved. The trademarks mentioned herein are the property of Life Technologies Corporation or their respective owners. TRIzol is a registered trademark of Molecular Research Center, Inc.