TRI REAGENT® - RNA / DNA / PROTEIN
Cat. No. TR 118
Store at 4 - 25 C
TRI Reagent® is a complete and ready-to-use reagent for the isolation of total RNA or the simultaneous isolation of RNA, DNA and proteins from samples of human, animal, plant, yeast, bacterial and viral origin. TRI Reagent is the improved version of the popular single-step method of total RNA isolation (1, 2). This highly reliable technique performs well with small and large quantities of tissues or cultured cells and allows simultaneous processing of a large number of samples. TRI Reagent and the single-step method are subjects of the international patents.
TRI Reagent combines phenol and guanidine thiocyanate in a mono-phase
solution to facilitate the immediate and most effective inhibition of RNase activity. A
biological sample is homogenized or lysed in TRI Reagent and the homogenate is separated
into aqueous and organic phases by bromochloropropane (cat. no. BP 151)
or chloroform addition and centrifugation. RNA remains exclusively in the aqueous phase, DNA in the interphase and
proteins in the organic phase. RNA is precipitated from the aqueous phase by addition of
isopropanol, washed with ethanol and solubilized. DNA and proteins are sequentially
precipitated from the interphase and the organic phase with ethanol and isopropanol, washed
with ethanol and solubilized.
STABILITY: TRI Reagent is stable at 25 C for at
least two years from the date of purchase (3). In case of contact
: Immediately flush eyes or skin with a large amount of water for at least 15 min
and seek immediate medical attention.
SPECIAL HANDLING PRECAUTIONS:TRI Reagent contains a poison (phenol) and an irritant (guanidine thiocyanate). Causes burns. CAN BE FATAL. When working with TRI Reagent use gloves and eye protection (shield, safety goggles). Do not get on skin or clothing. Avoid breathing vapor. Read the warning note on the bottle and MSDS.
In case of contact
: Immediately flush eyes or skin with a large amount of water for at least 15 min
and seek immediate medical attention.
I. ISOLATION OF RNA
TRI Reagent is the most effective method of RNA isolation. It isolates a whole spectrum of RNA molecules rarely observed in RNA isolated by other methods. Typically, the column-based methods may artificially change the mRNA composition. The entire procedure can be completed in 1 h and the recovery of undegraded mRNAs is 30-150% greater than with other methods of RNA isolation. TRI Reagent isolates high quality RNA from diverse biological material, including animal and plant tissues rich in polysaccharides and proteoglycans. The isolated RNA can be used for northern analysis, dot blot hybridization, poly A+ selection, in vitro translation, RNase protection assay, molecular cloning and RT-PCR. Simultaneous extraction of nearly 100% of the genomic DNA allows for normalization of the results of gene expression studies per genomic DNA instead of the more variable total RNA or tissue weight.
Reagents required, but not supplied: chloroform or bromochloropropane, isopropanol and ethanol.
We recommend the use of disposable polypropylene tubes (cat. no. PP 141-144) provided by Molecular Research Center, Inc. Tubes from other suppliers should be tested to ensure integrity during centrifugation at 12,000 g with TRI REAGENT.
1. HOMOGENIZATION: 1 ml TRI REAGENT + 50-100 mg tissue, 5-10 x 106 cells or 10 cm2 of culture plate.
2. PHASE SEPARATION: homogenate + 0.1 ml BCP or 0.2 ml
3. RNA PRECIPITATION: aqueous phase + 0.5 ml
4. RNA WASH: 1 ml 75% ethanol.
5. RNA SOLUBILIZATION: FORMAzol®, 0.5%
SDS, or water.
A. TISSUES. Homogenize tissue samples in TRI Reagent (1 ml/50 - 100 mg tissue) using a glass-Teflon or Polytron homogenizer. Sample volume should not exceed 10% of the volume of TRI Reagent used for homogenization.
B. CELLS. Cells grown in monolayer should be lysed directly in a culture dish. Pour off media, add TRI Reagent and pass the cell lysate several times through a pipette. Use 1 ml of TRI Reagnt per 10 cm2 of culture dish area. See also note #3 in Notes to the RNA isolation protocol. Cells grown in suspension should be sedimented first, and then lysed in TRI REAGENT by repetitive pipetting. Use 1.0 ml of the reagent per 5 - 10 x 106 animal, plant or yeast cells or per 107 bacterial cells.
Avoid washing cells before the addition of TRI Reagent as this may contribute to mRNA
degradation. Disruption of some yeast and bacterial cells may require the use of a
2. PHASE SEPARATION
Store the homogenate for 5 minutes at room temperature to permit the complete dissociation of nucleoprotein complexes. Next, supplement the homogenate with 0.1 ml BCP or 0.2 ml chloroform per 1 ml of TRI Reagent, cover the samples tightly and shake vigorously for 15 seconds. Store the resulting mixture at room temperature for 2-15 minutes and centrifuge at 12,000 g for 15 minutes at 4 C. Following centrifugation, the mixture separates into a lower red phenol-chloroform phase, interphase and the colorless upper aqueous phase. RNA remains exclusively in the aqueous phase whereas DNA and proteins are in the interphase and organic phase. The volume of the aqueous phase is about 60% of the volume of TRI Reagent used for homogenization.
Substituting BCP for chloroform does not affect the quality of isolated RNA, DNA or proteins and its use as the phase separation reagent may decrease the possiblity of contaminating RNA with DNA (4). Chloroform used for phase separation should not contain isoamyl alcohol or any other additive.
It is important to perform centrifugation to separate aqueous and organic phases in the cold ( 4-10 C ). If performed at elevated temperature, a residual amount of DNA may sequester in the aqueous phase. In this case, RNA can be used for northern analysis but it may not be suitable for PCR.
3. RNA PRECIPITATION
Transfer the aqueous phase to a fresh tube and save the interphase and organic phase at 4 C for subsequent isolation of DNA and proteins. Precipitate RNA from the aqueous phase by mixing with isopropanol. Use 0.5 ml of isopropanol per 1 ml of TRI Reagent used for the initial homogenization. Store samples at room temperature for 5-10 minutes and centrifuge at 12,000 g for 8 minutes at 4 - 25 C. RNA precipitate (often invisible before centrifugation) forms a gel-like or white pellet on the side and bottom of the tube.
When isolating RNA from sources rich in polysaccharides and proteoglycans, perform the
modified precipitation described in the Troubleshooting Guide.
4. RNA WASH
Remove the supernatant and wash the RNA pellet ( by vortexing) with 75% ethanol and subsequent centrifugation at 7,500 g for 5 minutes at 4 - 25 C. Add at least 1 ml of 75% ethanol per 1 ml TRI Reagent used for the initial homogenization.
If the RNA pellet accumulates on the side of the tube and has a tendency to float,
sediment the pellet at 12,000 g.
5. RNA SOLUBILIZATION
Remove the ethanol wash and briefly air-dry the RNA pellet for 3 - 5 min. It is important not to completely dry the RNA pellet as this will greatly decrease its solubility. Do not dry RNA by centrifugation under vacuum. Drying is not necessary for solubilization of RNA in FORMAzol® (stabilized formamide, cat. no. FO 121). Dissolve RNA in FORMAzol, water or 0.5% SDS by passing the solution a few times through a pipette tip and incubating for 10-15 minutes at 55-60 C. Water or the SDS solution used for RNA solubilization should be made RNase-free by diethyl pyrocarbonate (DEPC) treatment. RNA should be precipitated from FORMAzol with ethanol before using for RT-PCR.
TRI Reagent isolates a whole spectrum of RNA molecules rarely observed in RNA preparations isolated by other methods. Ethidium bromide staining of RNA separated in an agarose gel or methylene blue staining of a hybridization membrane after RNA transfer visualizes two predominant bands of small (~2 kb) and large (~5 kb) ribosomal RNA, low molecular weight (0.1-0.3 kb) RNA, and discrete bands of high molecular weight (7-15 kb) RNA.
The final preparation of total RNA is essentially free of DNA and proteins and has a 260/280 ratio 1.6 - 1.9. For RT-PCR analysis, DNase treatment may be necessary for optimal results. For optimal spectrophotometric measurements, RNA aliquots should be diluted with water or buffer with a pH > 7.5 such as Phosphate Buffer (cat. no. SP 130). Distilled water with a pH < 7.0 falsely decreases the 260/280 ratio and impedes the detection of protein contamination in RNA samples (7).
A) tissues (µg RNA/mg tissue): liver, spleen, 6-10 µg; kidney, 3-4 µg; skeletal muscles, brain, 1-1.5 µg; placenta, 1-4 µg;
B) cultured cells (µg RNA/106 cells): epithelial cells, 8-15 µg; fibroblasts, 5-7 µg.
II. ISOLATION OF DNA BY TRI REAGENT
The DNA is isolated from the interphase and phenol phase separated from the initial
homogenate as described in the RNA isolation protocol. Following precipitation and a
series of washes, the DNA is solubilized in 8 mM NaOH, neutralized and used for analysis.
The DNA isolated by TRI Reagent can be used for PCR, restriction digestion and Southern
blotting. In addition, full recovery of DNA from tissues and cultured cells permits the
use of TRI Reagent for determination of the DNA content in analyzed samples (2).
Reagents required, but not supplied: ethanol, trisodium citrate and sodium hydroxide.
1. DNA PRECIPITATION: phenol phase and interphase + 0.3 ml ethanol (1 ml TRI
2. DNA WASH: 1 ml 0.1 M trisodium citrate in 10% ethanol x 2; 2 ml 75% ethanol.
3. DNA SOLUBILIZATION: 8 mM NaOH.
The procedure is carried out at room temperature, unless stated otherwise.
1. DNA PRECIPITATION
Remove the remaining aqueous phase overlying the interphase. Precipitate DNA from the interphase and organic phase with ethanol. Add 0.3 ml of 100% ethanol per 1 ml of TRI Reagent used for the initial homogenization, and mix samples by inversion. Next, store the samples at room temperature for 2-3 minutes and sediment DNA by centrifugation at 2,000 g for 5 minutes at 4 C. Careful removal of any residual aqueous phase is critical for the quality of the isolated DNA.
See Notes #6 for an alternative DNA isolation procedure.
2. DNA WASH
Remove the phenol-ethanol supernatant and store at 4 C for the subsequent protein isolation. Wash the DNA pellet twice in a solution containing 0.1 M trisodium citrate in 10% ethanol. (No pH adjustment required.) Use 1 ml of the solution per 1 ml of TRI Reagent used for the initial homogenization. At each wash, store the DNA pellet in the washing solution for 30 minutes at room temperature with periodic mixing and centrifuge at 2,000 g for 5 minutes at 4 - 25 C. Next, suspend the DNA pellet in 75% ethanol (1.5-2 ml of 75% ethanol per 1 ml TRI REAGENT), store for 10 - 20 min at room temperature with periodic mixing and centrifuge at 2,000 g for 5 minutes at 4-25 C. This ethanol wash removes pinkish color from the DNA pellet.
An additional wash in 0.1M trisodium citrate-10% ethanol is required for large pellets containing >200 µg DNA or large amounts of a non-DNA material.
3. DNA SOLUBILIZATION
Remove the ethanol wash and briefly air-dry the DNA pellet by keeping tubes open for 3 - 5 minutes at room temperature. Dissolve the DNA pellet in 8 mM NaOH by slowly passing through a pipette. Add an adequate amount of 8 mM NaOH to approach a DNA concentration of 0.2 - 0.3 µg/µl. Typically, add 0.3 - 0.6 ml 8 mM NaOH to DNA isolated from 50 - 70 mg of tissue or 107 cells. The use of a mild alkaline solution assures full solubilization of the DNA pellet. At this stage, the DNA preparations (especially from tissues) still contain insoluble material (fragments of membranes,etc.); remove this material by centrifugation at 12,000 g for 10 minutes and transfer the resulting supernatant containing DNA to new tubes. A high viscosity of the supernatant indicates the presence of high molecular weight DNA.
QUANTITATION OF DNA
For optimal spectrophotometric measurements, DNA aliquots should be diluted with water or buffer with a pH >7.5 such as Phosphate Buffer (cat. no. SP 130). Distilled water with a pH < 7.0 falsely decreases the 260/280 ratio and impedes the detection of protein in RNA samples (7).
Calculate the DNA content assuming that one A260 unit equals 50 µg of double-stranded DNA/ml. For calculation of the cell number in analyzed samples, assume that the amount of DNA per 106 of diploid cells of human, rat and mouse origin equals 7.1 µg, 6.5 µg and 5.8 µg, respectively (5). A typical preparation of DNA isolated from tissues is composed of 60-100 kb DNA (70%) and ~20 kb DNA (30%). Preparations isolated from cultured cells contain >80% of 60-100 kb DNA and <20% ~20 kb DNA. The isolated DNA is free of RNA and proteins and has a 260/280 ratio >1.7.
Expected yield: A) Tissues (µg DNA/mg tissue): liver, kidney, 3-4 µg: skeletal muscles, brain, placenta 2-3 µg.
B) Cultured human, rat and mouse cells 5-7 ug DNA/106 cells.
AMPLIFICATION OF DNA BY PCR
Following solubilization in 8 mM NaOH, adjust the pH of the DNA sample to 8.4 using HEPES (see Table). Add an aliquot of the sample (typically 0.1 - 1 µg DNA) to a PCR reaction mix and perform PCR according to your standard protocol.
DIGESTION OF DNA BY RESTRICTASES
Adjust the pH of the DNA solution to a required value using HEPES (see Table). Alternatively, dialyze samples against 1 mM EDTA, pH 7 - pH 8. Carry out the DNA restriction for 3 - 24 h under optimal conditions for a specific restrictase using 3-5 units of the enzyme per µg DNA. In a typical assay, 80-100% of the DNA preparation is digested by restrictases.
III. ISOLATION OF PROTEINS BY TRI REAGENT
Revised protocol reduces extraction time and improves protein recovery!
Proteins are isolated from the phenol-ethanol supernatant obtained after precipitation of DNA with ethanol (step 1, DNA PRECIPITATION). The resulting preparation can be analyzed for the presence of specific proteins by Western blotting (2,8,9).
Reagents required but not supplied: guanidine hydrochloride, ethanol, isopropanol, acetone, glycerol, SDS, urea and tributylphosphine (Sigma T 7567).
The protocol includes the following steps:
1. PROTEIN PRECIPITATION - 0.2-0.5 ml phenol-ethanol supernatant (1 volume) + acetone (3 volumes)
2. PROTEIN WASH - 1 ml of guanidine hydrochloride/ethanol/glycerol wash solution, 3 x 10 min.
- 1 ml ethanol/glycerol solution, 1 x 10 min.
3. PROTEIN SOLUBILIZATION - 1% SDS, 10M Urea or other suitable solvent.
The procedure is carried out at room temperature unless stated otherwise.
1. PROTEIN PRECIPITATION
Aliquot a portion of the phenol-ethanol supernatant (0.2 - 0.5 ml, 1 volume) into a microfuge tube. Precipitate proteins by adding 3 volumes of acetone. Mix by inversion for 10-15 sec to obtain a homogeneous solution. Store samples for 10 min at room temperature and sediment the protein precipitate at 12,000 g for 10 min at 4 C (See Notes 1and 2).
2. PROTEIN WASH
Decant the phenol-ethanol supernatant and disperse the protein pellet in 0.5 ml of 0.3 M guanidine hydrochloride in 95% ethanol + 2.5 % glycerol (V:V). Disperse the pellet using a pipet tip, syringe needle or a small conical Teflon pestle (Fisher K749515-0000) attached to a mechanical stirrer (~30 sec @ 800-1000 RPM). After dispersing the pellet, add another 0.5 ml aliquot of the guanidine hydrochloride/ethanol/glycerol wash solution to the sample and store for 10 min at RT. Sediment the protein at 8,000 g for 5 min. Decant the wash solution and perform two more washes in 1 ml each of the guanidine/ethanol/glycerol wash solution. Disperse the pellet by vortexing after each wash to efficiently remove residual phenol. Perform the final wash in 1 ml of ethanol containing 2.5 % glycerol (V:V). At the end of the 10 min ethanol wash, sediment the protein at 8,000 g for 5 minutes. Decant the alcohol, invert the tube and dry the pellet for 7-10 min at room temperature (See Note 3).
3. PROTEIN SOLUBILIZATION
Option 1. After briefly air-drying the protein pellet, add a suitable solvent such as 1% SDS, 10 M urea, or another suitable detergent-based solvent to the protein pellet (9). Use 0.2 ml of solvent per 10-20 mg of tissue sample (See Note 4). Gently disperse and solubilize the pellet for 15-20 minutes by "flicking" the tube or pipetting as required. The addition of a suitable reducing agent such as tributylphosphine (2.5% of solution volume) will improve protein yield in most preparations. For immediate use in western analysis, heat the solution for 3 min at 100 C and sediment any insoluble material by centrifugation at 10,000 g for 5 min at RT. Transfer the supernatant to a clean tube and use the protein solution immediately for western blotting (See Note 5). Otherwise, store the solubilized proteins at -20 C and perform the heating, centrifugation or other preparatory steps at the time of use.
Option 2. Dialyze the phenol-ethanol supernatant (II DNA Isolation Step 1, DNA Precipitate) in a suitable, regenerated cellulose dialysis tubing against three changes of 0.1% SDS at 4 C. Centrifuge the dialysate at 10,000 g for 10 min at 4 C and use the clear supernatant for Western blotting.
NOTES1. Isopropanol may replace acetone during protein precipitation but total recovered protein yield may be reduced by 5-10 % (8).
2. Limiting the volume of phenol - ethanol supernatant to 0.2 - 0.5 ml per tube will produce a smaller, more manageable protein pellet and improve protein yield. TRI Reagent protein extracts prepared from rat tissues yield 50 - 110 µg protein / mg tissue.
3. In general, protein pellets suspended in 0.3 M guanidine hydrochloride/ethanol/glycerol wash solution or in ethanol/glycerol wash solution can be stored for at least one month at 4 C or one year -20 C. Individual proteins may display different sensitivity to long-term storage and optimal storage conditions should be established for sensitive and labile proteins.
4. The solubility and stability of specific proteins can be influenced by different detergent solutions (9). To obtain optimal results in various experimental applications, investigators may solubilize small amounts of protein in different solvents and determine which solution best addresses their unique experimental objectives.
5. Solubilized protein may form insoluble aggregates during storage at -20 C. Prior to western analysis, thaw the samples at 25 C for 10-15 minutes. Heat the solubilized protein sample for 3 min at 100 C, pipette the solution and remove insoluble protein by centrifugation as outlined in the protocol.
IV. TROUBLESHOOTING GUIDERNA ISOLATION
V. ISOLATION OF POLY A+ RNA
Following RNA precipitation with isopropanol (step 3), the RNA pellet
can be dissolved in a poly A+ binding buffer and poly A+ RNA
selection can be performed on an oligo-dT column or using any commercial product
according to a standard protocol of Aviv and Leder (Proc Natl Acad Sci USA, 1972, 69, 1408-1412).
VI. RT-PCR APPLICATION NOTES
The additional centrifugation described in Notes #5 to the RNA
isolation protocol further eliminates the possibility of DNA contamination in
RNA extracted by TRI Reagent.
A more complete evaporation of ethanol is required for RNA samples used in RT-PCR. This is especially important for small volume samples (5-20 µl) which, if not dried sufficiently, may contain a relatively high level of ethanol.
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Last modified: July 17, 2012