TRIzol™ Reagent
Catalog Numbers 15596026 and 15596018
Doc. Part No. 15596026.PPS Pub. No. MAN0001271 Rev.
A.0
WARNING! Read the Safety Data Sheets (SDSs) and follow the
handling instructions. Wear appropriate protective eyewear,
clothing, and gloves. Safety Data Sheets (SDSs) are available
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Product information
Invitrogen™ TRIzol™ Reagent is a ready-to-u 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 RNa 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 and Sacchi, 1987).
TRIzol™ Reagent allows to perform quential 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 parate into a clear upper aqueous layer (containing RNA), an interpha, and a red lower organic layer (containing the DNA and proteins). R
NA is precipitated from the aqueous layer with isopropanol. DNA is precipitated from the interpha/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 u in downstream applications.•Isolated RNA can be ud in RT-PCR, Northern Blot analysis, Dot Blot hybridization, poly(A)+ lection, in vitro translation, RNa protection assay, and molecular cloning.
•Isolated DNA can be ud in PCR, Restriction Enzyme digestion, and Southern Blots.
•Isolated protein can be ud for Western Blots, recovery of some enzymatic activity, and some immunoprecipitation.
TRIzol™ Reagent can also be ud with Phamaker™ Tubes to isolate RNA. Refer to TRIzol™ Reagent and Phamaker™ Tubes Complete System Ur Guide (MAN0016163) for the full protocol.
Contents and storage
Required materials not supplied
Unless otherwi indicated, all materials are available MLS: Fisher Scientific () or other major laboratory supplier.
Table 1 Materials required for RNA, DNA, and protein isolation
Table 2 Materials required for RNA isolation
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Table 3 Materials required for DNA isolation
Table 4
Materials required for protein isolation
Input sample requirements
IMPORTANT! Perform RNA isolation immediately after sample collection or quick-freeze samples immediately after collection and store at –80°C or in liquid nitrogen until RNA isolation.
Fresh tissues or tissues stored in RNA™ Stabilization Solution (Cat.
No. AM7020).
Procedural guidelines
•Perform all steps at room temperature (20–25°C) unless otherwi noted.
•U cold TRIzol™ Reagent if the starting material contains high levels of RNa, such as spleen or pancreas samples.
•U disposable, individually wrapped, sterile plastic ware and
sterile, disposable RNa-free pipettes,pipette tips, and tubes.•Wear disposable gloves while handling reagents and RNA
samples to prevent RNa contamination from the surface of the skin; change gloves frequently, particularly as the protocol
progress from crude extracts to more purified materials.•Always u proper microbiological aptic techniques when
working with RNA.
•U RNa Zap™ RNa Decontamination Solution (Cat.
no. AM9780) to remove RNa contamination from work surfaces and non-disposable items such as centrifuges and pipettes ud during purification.
Ly samples and parate phas
出席英语1.Ly and homogenize samples in TRIzol™ Reagent according to
your starting material.
•Tissues:
Add 1 mL of TRIzol™ Reagent per 50–100 mg of tissue to the
sample and homogenize using a homogenizer.
•Cell grown in monolayer:
a.Remove growth media.
b.Add 0.3–0.4 mL of TRIzol™ Reagent per 1 × 105—107 cells
directly to the culture dish to ly the cells.
c.Pipet the lysate up and down veral times to homogenize.
•Cells grown in suspension:
a.Pellet the cells by centrifugation and discard the
supernatant.
b.Add 0.75 mL of TRIzol™ Reagent per 0.25 mL of sample (5–
10 × 106 cells from animal, plant, or yeasty origin or 1 ×107
cells of bacterial origin) to the pellet.
Note: Do not wash cells before addition of TRIzol™ Reagent
to avoid mRNA degradation.
c.Pipet the lysate up and down veral times to homogenize.
Note: The sample volume should not exceed 10% of the volume of TRIzol™ Reagent ud for lysis.
参观的英文STOPPING POINT Samples can be stored at 4°C overnight or at –
20°C for up to a year.
2.(Optional) If samples have a high fat content, centrifuge the lysate
for 5 minutes at 12,000 × g at 4–10°C, then transfer the clear
supernatant to a new tube.
3.Incubate for 5 minutes to permit complete dissociation of the
nucleoproteins complex.
4.Add 0.2 mL of chloroform per 1 mL of TRIzol™ Reagent ud for
lysis, then curely cap the tube.
5.Incubate for 2–3 minutes.
6.Centrifuge the sample for 15 minutes at 12,000 × g at 4°C.
The mixture parates into a lower red phenol-chloroform, and
interpha, and a colorless upper aqueous pha.
7.Transfer the aqueous pha containing the RNA to a new tube.
8.Transfer the aqueous pha containing the RNA to a new tube by
angling the tube at 45° and pipetting the solution out.
IMPORTANT! Avoid transferring any of the interpha or organic layer into the pipette when removing the aqueous pha. Proceed directly to “Isolate RNA“ on page 2.
Save the interpha and organic pha if you want to isolate DNA or protein. See “Isolate DNA“ on page 3 or “Isolate proteins“ on
page 4 for detailed procedures. The organic pha can be stored at 4°C overnight.
Isolate RNA
a.(Optional) If the starting sample is small (<106 cells or <10 mg of tissue), add 5–10 µg of RNa-free
glycogen as a carrier to the aqueous pha.
Note: The glycogen is co-precipitated with the RNA, but does not interfere with subquent
applications.
b.Add 0.5 mL of isopropanol to the aqueous pha, per 1 mL of TRIzol™ Reagent ud for lysis.
c.Incubate for 10 minutes.
d.Centrifuge for 10 minutes at 12,000 × g at 4°C.
Total RNA precipitate forms a white gel-like pellet at the bottom of the tube.
e.Discard the supernatant with a micropipettor.
1Precipitate the RNA
a.Resuspend the pellet in 1 mL of 75% ethanol per 1 mL of TRIzol™ Reagent ud for lysis.
Note: The RNA can be stored in 75% ethanol for at least 1 year at –20°C, or at least 1 week at 4°C.
b.Vortex the sample briefly, then centrifuge for 5 minutes at 7500 × g at 4°C.
c.Discard the supernatant with a micropipettor.
d.Vacuum or air dry the RNA pellet for 5–10 minutes.
IMPORTANT! Do not dry the pellet by vacuum centrifuge. Do not let the RNA pellet dry, to ensure total
solubilization of the RNA. Partially dissolved RNA samples have an A230/280 ratio <1.6.
2Wash the RNA
3Solubilize the RNA
a.Resuspend the pellet in 20–50 µL of RNa-free water, 0.1 mM EDTA, or 0.5% SDS solution by pipetting
up and down.
IMPORTANT! Do not dissolve the RNA in 0.5% SDS if the RNA is to be ud in subquent enzymatic
reactions.
b.Incubate in a water bath or heat block t at 55–60°C for 10–15 minutes.
Proceed to downstream applications, or store the RNA at –70°C.
4Determine the RNA yield
Table 5 Typical RNA (A260/280 of >1.8) yields from various starting materials
Isolate DNA from the interpha and the lower phenol-chloroform pha saved from “Ly samples and parate phas“ on page 2.
1Precipitate the DNA
a.Remove any remaining aqueous pha overlying the interpha.
This is critical for the quality of the isolated DNA.
b.Add 0.3 mL of 100% ethanol per 1 mL of TRIzol™ Reagent ud for lysis.
c.Cap the tube, mix by inverting the tube veral times.
d.Incubate for 2–3 minutes.
e.Centrifuge for 5 minutes at 2000 × g at 4°C to pellet the DNA.
f.Transfer the phenol-ethanol supernatant to a new tube.
The supernatant is ud for protein isolation (e “Isolate proteins“ on page 40, if needed, and can be
stored at –70°C for veral months.
2Wash the DNA
a.Resuspend the pellet in 1 mL of 0.1 M sodium citrate in 10% ethanol, pH 8.5, per 1 mL of TRIzol™
Reagent ud for lysis.
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b.Incubate for 30 minutes, mixing occasionally by gentle inversion.
Note: The DNA can be stored in sodium citrate/ethanol for at least 2 hours.
c.Centrifuge for 5 minutes at 2000 × g at 4°C.
d.Discard the supernatant with a micropipettor.
e.Repeat step 2a–step 2d once.
数学真好玩Note: Repeat step 2a–step 2d twice for large DNA pellets (>200 µg).
f.Resuspend the pellet in 1.5–2 mL of 75% ethanol per 1 mL of TRIzol™ Reagent ud for lysis.
g.Incubate for 10–20 minutes, mixing occasionally by gentle inversion.
Note: The DNA can be stored in 75% ethanol at veral months at 4°C.
h.Centrifuge for 5 minutes at 2000 × g at 4°C.
i.Discard the supernatant with a micropipettor.
j.Vacuum or air dry the DNA pellet for 5–10 minutes.
IMPORTANT! Do not dry the pellet by vacuum centrifuge.
3Solubilize the DNA
a.Resuspend the pellet in 0.3–0.6 mL of 8 mM NaOH by pipetting up and down.
Note: We recommend resuspending the DNA is a mild ba becau isolated DNA does not resuspend
well in water or Tris buffer.
b.Centrifuge for 10 minutes at 12,000 × g at 4°C to remove insoluble materials.
c.Transfer the supernatant to a new tube, then adjust pH as needed with HEPES.
Proceed to downstream applications, or store the DNA at 4°C overnight. For longer-term storage at –20°C,
adjust the pH to 7–8 with HEPES and add 1 mM EDTA.
4Determine the DNA yield
Table 6 Typical DNA (A260/280 of 1.6–1.8) yields from various starting materials
Isolate the proteins from the phenol-ethanol supernatant saved from “Precipitate the DNA“ on page 3 using either “Precipitate the proteins“ on page 4 or “Dialy the proteins“ on page 5.
1Precipitate the proteins
a.Add 1.5 mL of isopropanol to the phenol-ethanol supernatant per 1 mL of TRIzol™ Reagent ud for
lysis.
b.Incubate for 10 minutes.
c.Centrifuge for 10 minutes at 12,000 × g at 4°C to pellet the proteins.
d.Discard the supernatant with a micropipettor.
2Wash the proteins
a.Prepare a wash solution consisting of 0.3 M guanidine hydrochloride in 95% ethanol.
b.Resuspend the pellet in 2 mL of wash solution per 1 mL of TRIzol™ Reagent ud for lysis.
c.Incubate for 20 minutes.
Note: The proteins can be stored in wash solution for at least 1 month at 4°C or for at least 1 year at –
20°C.
d.Centrifuge for 5 minutes at 7500 × g at 4°C.
e.Discard the supernatant with a micropipettor.
f.Repeat step 2b–step 2e twice.
g.Add 2 mL of 100% ethanol, then mix by vortexing briefly.
h.Incubate for 20 minutes.
集锦的意思i.Centrifuge for 5 minutes at 7500 × g at 4°C.
j.Discard the supernatant with a micropipettor.
k.Air dry the protein pellet for 5–10 minutes.
IMPORTANT! Do not dry the pellet by vacuum centrifuge.
3Solubilize the proteins
a.Resuspend the pellet in 200 µL of 1% SDS by pipetting up and down.
Note: To ensure complete resuspension of the pellet, we recommend that you incubate the sample at
50°C in a water bath or heat block.
b.Centrifuge for 10 minutes at 10,000 × g at 4°C to remove insoluble materials.
c.Transfer the supernatant to a new tube.
Proceed directly to downstream applications, or store the sample at –20°C.
•Measure protein concentration by Bradford assay.
Note: SDS concentration mush be <0.1%.
4Determine the protein yield
Dialy the proteins
1.Load the phenol-ethanol supernatant into the dialysis membrane.
Note: The phenol-ethanol solution can dissolve some types of
dialysis membranes (cellulo ester, for example). Test dialysis
tubing with the membrane to asss compatibility before starting.
2.Dialyze the sample against 3 changes of 0.1% SDS at 4°C. Make
the first change of solution after 16 hours, the cond change 4
hours later (at 20 hours), and the final change 2 hours later (at 22 hours).
Note: A SDS concentration of at least 0.1% is required to
resolubilize the proteins from the pellet. If desired, the SDS can be diluted after solubilization.
3.Centrifuge the dialysate for 10 minutes at 10,000 × g at 4°C.
4.Transfer the supernatant containing the proteins to a new tube.
5.(Optional) Solubilize the pellet by adding 100 µL of 1% SDS and
100 µL of 8 M urea.
Proceed directly to downstream applications, or store the sample at –20°C.
Troubleshooting
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雪花的诗句References
Chomczynski, P. 1993. A reagent for the single-step simultaneous isolation of RNA, DNA and proteins from cell and tissue samples. BioTechniques 15, 532-537Chomczynski, 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., and 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
