TRIzol™ LS Reagent

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We recommend using straight chloroform. No isoamyl alcohol is needed (though using chloroform:isoamyl alcohol 49:1 works without problems). You can also use chloroform with 50 ppm amylene. Alternatively, BCP (1-bromo-2 chloropropane) can be used in the place of chloroform.

Find additional tips, troubleshooting help, and resources within our RNA Sample Collection, Protection, and Isolation Support Center.

Small volumes (0.5-0.8 mL) of TRIzol Reagent have been used successfully for 10^2 to 10^5 cells, but if small volumes are used, we recommend using smaller tubes in order to have the tallest possible column of aqueous phase. The taller the column of liquid, the less likely that contamination from the interphase will occur.

Here is a protocol for isolation of RNA from small quantities of tissue (1-10 mg) or cells (100-10,000):
1. Add 800 µL TRIzol Reagent to the sample. Homogenize cells by pipetting repeatedly. Add 200 µg glycogen (Cat. No. 10814010) directly to the TRIzol Reagent. If processing tissue, pulverize in liquid nitrogen first and then add 800 µL TRIzol Reagent containing 200 µg glycogen (final concentration 250 µg/mL) followed by vigorous vortexing or power homogenization.
2. Place at room temperature, cap the vial, and vortex at high speed for 10 seconds. Make sure the TRIzol Reagent wets the side of the vial in order to solubilize any sample that may be remaining on the walls.
3. Shear the genomic DNA in the sample by passing twice through a 26-gauge needle connected to a 1 mL syringe. Using the syringe, transfer the sample to a sterile 1.5 mL microcentrifuge tube.
4. Add 160 µL of chloroform (or 49:1 chloroform:isoamyl alcohol) to each sample and vortex up to 30 seconds. Centrifuge at maximum speed in a microcentrifuge for 5 minutes to separate the phases.
5. Transfer the upper aqueous phase to a fresh tube and add 400 µL ice-cold isopropanol. Allow the samples to precipitate at -20 degrees C for 1 hour to overnight. Pellet the RNA by centrifugation at maximum speed in the microfuge for 15 minutes at room temperature.
6. Decant the supernatant. Wash the pellet in 200 µL of 70% ethanol and centrifuge again for 10 minutes at maximum speed. Decant the supernatant, removing as much as possible without disturbing the pellet. Dry the RNA pellet.
7. Resolubilize the pellet in 30-50 µL RNAse-free deionized water. If tissue is high in RNAses (e.g., adrenal gland, pancreas), resuspend in 100% deionized formamide. Be sure to vortex or pipette the sample up and down to ensure that the pellet is fully resolubilized. Store at -70 degrees C.

Find additional tips, troubleshooting help, and resources within ourRNA Sample Collection, Protection, and Isolation Support Center.

There are several possible stopping points and recommended storage conditions during the extraction of RNA with TRIzol Reagent:

- Sample homogenization step: After homogenization (before addition of chloroform), you can store samples at -70 degrees C for at least 1 year. The homogenated samples can sit at room temperature for several hours before adding chloroform.
- Sample homogenization step: If samples are efficiently lysed in TRIzol Reagent and the reagent inactivates the nucleases, you can safely store RNA for 3-4 days at room temperature.
- RNA precipitation step: You can store RNA in isopropanol overnight at 4 degrees C. Prolonged storage at this reduced temperature will not influence the yield of RNA appreciably. Do not store at -20 degrees C, as salts will precipitate, and do not store for a prolonged time at room temperature because the guanidine isothiocyanate can harm the RNA.
- RNA wash step: You can store RNA in 75% ethanol for 1 week at 4 degrees C or 1 year at -20 degrees C.

Find additional tips, troubleshooting help, and resources within our RNA Sample Collection, Protection, and Isolation Support Center.

The absorbance of nucleic acids is dependent upon the ionic strength and pH of the medium. Please see the range of absorbance values below based on the diluents used.

Diluent A260 A280 A260/A280 RNA (µg/mL)
Cytoplasmic RNA dissolved in distilled water 0.381 0.223 1.711 15.24
Cytoplasmic RNA dissolved in TE buffer 0.335 0.145 2.310 13.4
RNA isolated by TRIzol Reagent and dissolved in distilled water 0.585 0.328 1.785 23.4 RNA isolated by TRIzol Reagent and dissolved in TE buffer 0.544 0.247 2.206 21.76 Although a high A260/A280 ratio may not indicate an extremely pure preparation of nucleic acid, a low A260/A280 ratio (1.7 for RNA) does indicate that the preparation is contaminated and may not be suitable for some applications.

Find additional tips, troubleshooting help, and resources within our RNA Sample Collection, Protection, and Isolation Support Center.