Manual Maquina Hielo

TRI REAGENT® - RNA / DNA / PROTEIN ISOLATION REAGENT Cat. No. TR 118 Store at 4 - 25 C

PRODUCT DESCRIPTION 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). 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.

PROTOCOL 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.

The protocol includes the following steps:

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 chloroform.

3. RNA PRECIPITATION: aqueous phase + 0.5 ml isopropanol.

4. RNA WASH: 1 ml 75% ethanol.

5. RNA SOLUBILIZATION: FORMAzol®, 0.5% SDS, or water.

The procedure is performed at room temperature,unless stated otherwise.

1. HOMOGENIZATION 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 homogenizer.

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.

6. RESULTS 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).

Expected yield: 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.

NOTES

1. To facilitate isolation of RNA from small samples (<106 cells or <10 mg tissue) perform homogenization (or lysis) in 0.8 ml of TRI Reagent supplemented with 2 - 8 μl of Polyacryl Carrier (cat. no. PC 152). Next, add BCP or chloroform and proceed with the phase separation and other steps of isolation as described above.

2. After homogenization (before addition of chloroform) samples can be stored at -70 C for at least one month. The RNA precipitate (step 4, RNA WASH) can be stored in 75% ethanol at 4 C for at least one week or at least one year at -20 C.

3. For cells grown in monolayer, use the amount of TRI Reagent based on the area of a culture dish and not on cell number. The use of an insufficient amount of TRI Reagent may result in contamination of the isolated RNA with DNA.

4. Hands and dust may be a major source of the RNase contamination. Use gloves and keep tubes

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