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. TRlzol® Reagent is a monophasic solution of
phenol, guanidine isothiocyanate, and other proprietary components which
facilitate the isolation of a variety of RNA spedes of
large or small molecular size. TRIzol® Reagent maintains the integrity
of the RNA due to higMy 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 précipitation 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 preipitation. 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.
RNA Isolation Procedure
Always use the appropriate precautions to avoid RNase contamination when
preparing and handling RNA.
RNA précipitation
l. (Optional) When precipitating RNA from small sample
quantities (<=10^6 cells or <10 mg tissue), add 5-10 µg 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.
2. Add 0.5 mL of 100% isopropanol to the aqueous phase, per
l m L of TRIzol® Reagent used for homogenization.
3. Incubate at room temperature for 10 minutes.
4. Centrifuge at 12/000 x g for 10 minutes at 4°C.
Note: The RNA is often invisible prior to centrifugation, and forms a
gel-like pellet on the side and bottom of the
tube.
5. Proceed to RNA wash.
RNA wash
1. Remove the supenatant from the tube, leaving only the
RNA pellet.
2. Wash the pellet, with 1 mL of 75% ethanol per l mL of
TRIzol® Reagent used in the initial homogenization.
Note: The RNA can be stored in 75% ethanol at least l year at -20°C/ or at least 1 week at 4°C.
3. Vortex the sample briefly, then centrifuge the tube at
7500 x g for 5 minutes at 4°C. Discard the wash.
4. Vacuum or air dry the RNA pellet for 5-10 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.
5. Proceed to RNA resuspension.
RNA resuspension
l. Resuspend the RNA pellet in RNase-free water or
0.5% SOS solution (20-50 yL) 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.
2. Incubate in a water bath or heat block set at 55-60°C for
10-15 minutes.
3. Proceed to downstream application, or store at -70°C
Aucun commentaire:
Enregistrer un commentaire