Tri Pure Isolation Reagent
Tri Pure Isolation Reagent
Tri Pure Isolation Reagent
Starting material
Preparation of total RNA, genomic DNA, and protein from a single biological sample
DNA-free total RNA may be used for Northern blots, in vitro translation, RNase
protection assays, cDNA synthesis, or RT-PCR
RNA-free DNA may be used for PCR, restriction analysis, Southern blots, and
cloning
Denatured protein may be used for SDS-PAGE and Western blots
Time required
Results
Yields vary depending on starting material (See the table under Part IV of How to
use the reagent in this article)
A260/A280 of RNA = 1.6 2.0
A260/A280 of DNA >1.7
Benets
156
Flow diagram
(see page 159)
White interphase
Red organic phase
II.
Contain DNA
and protein
Reagent contents
TriPure Isolation Reagent is a clear, red, monophasic solution of phenol and guanidine
thiocyanate, pH 4. It is ready to use as supplied.
III.
Sterile, disposable polypropylene tubes that can withstand 12,000 x g in the presence
of TriPure Isolation Reagent and chloroform
Chloroform (free of all additives such as isoamyl alcohol)
For RNA isolation
Isopropanol
75% ethanol
Diethylpyrocarbonate (DEPC)-treated, RNase-free water or DEPC-treated 0.5% SDS
For DNA isolation
Absolute ethanol
75 % ethanol
8 mM NaOH
0.1 M sodium citrate in 10 % ethanol
For protein isolation
Isopropanol
1 % sodium dodecyl sulfate (SDS)
0.3 M guanidine hydrochloride (GuHCl) in 95% ethanol
Absolute ethanol
Solution-based Isolation
157
IV.
158
Sample
RNA yield
DNA yield
Tissue:
Liver
Spleen
Kidney
Skeletal muscle or brain
Placenta
6 10 g/mg tissue
6 10 g/mg tissue
3 4 g/mg tissue
1.0 1.5 g/mg tissue
1 4 g/mg tissue
3 4 g/mg tissue
not determined
3 4 g/mg tissue
2 3 g/mg tissue
2 3 g/mg tissue
Cultured cells:
Epithelial cells
Fibroblasts
Human, mouse, or rat cells
8 15 g/106 cells
5 7 g/106 cells
not determined
not determined
not determined
5 7 g/106 cells
V.
Isolation of RNA
Isolation of DNA/protein
Precipitate with
isopropanol
(0.5 ml per 1 ml TriPure)
Mix by inversion
Incubate 5 10 min at
15 to 25C
Centrifuge 12,000 x g,
10 min at 2 to 8C
Discard suppernatant
Centrifuge 7500 x g,
5 min at 2 to 8C
Discard supernatant
Solution-based Isolation
Isolation of DNA
Isolation of protein
Pellet
Phenol/EtOH supernatant
159
Store RNA at 15
to 25C
Centrifuge 2000 x g,
5 min at 2 to 8C
Discard supernatant
Centrifuge 12,000 x g,
10 min at 2 to 8C
Discard supernatant
Resuspend pellet in
0.3 M GuHCl in 95% EtOH
(2 ml per 1 ml TriPure)
Incubate pellet in wash 20 min
at 15 to 25C
Store DNA at 2 to 8C
160
V.
During
If you get...
A260/A280 ratio
<1.65
Solution-based Isolation
161
V.
During
If you get...
A260/A280 ratio
<1.7
DNA
degradation
RNA
contamination
Low protein
yield
Protein
degradation
Deformed
bands in PAGE
analysis
162
10
11
12
13
Figure 44: Northern blot with total RNA isolated by the TriPure Isolation Reagent. Total RNA was isolated
(by the TriPure protocol) from the following research samples: 1.5 x 106 cells of a human leukemia cell line, 5.0 x
107 human white blood cells, 1.7 x 107 buffy coat cells from human blood, and 500 mg rat tissue. The isolated
RNA samples were separated electrophoretically on a gel, transferred to a nylon membrane, and hybridized with
a 1 kb, digoxigenin-labeled glyceraldehyde 3-phosphate dehydrogenase (G3PDH) probe. The blot was incubated
overnight with DIG System reagents for chemiluminescent detection, then exposed to X-ray lm for 5 min. The
G3PDH probe recognizes a 1.35 kb mRNA, as shown in:
Lane 1: RNA ladder
Lanes 2 4: RNA from human leukemia cell line
Lanes 5 7: RNA from human white blood cell pellet
Lanes 8 10: RNA from human blood buffy coat
Lanes 11 13: RNA from rat liver tissue
The amount of total RNA applied to the original gel was either 5 g (lanes 2, 5, 8, 11), 1 g (lanes 3, 6, 9, 12), or
0.25 g (lanes 4, 7, 10, 13).
Result: This data clearly demonstrates that high quality, intact RNA is successfully isolated from a variety of
starting materials using the TriPure Isolation Reagent.
References
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Bakker, O. (1998) Biochemica 1, 22-23; Biochemica Information (1998) 102, 30 31
Bystriansky, J. S. and Ballantyne, J. S. (2007) Am J Physiol Regulatory Integrative Comp
Physiol, 292, R1043 1051
Chomczynski, P. (1993) BioTechniques 15, 532 537
Garry D. et al. (1996) Nature 395, 905 908
Lande-Diner, L. et al. (2007) J. Biol. Chem., 282, 12194 12200
Morris, T. et al. (1996) J. Clin. Microbiology 2933 2936
Naseem, R. H. et al. (2007) Physiol Genomics, 30, 44 52
Schummer, B. et al. (1998) Technical Tip Biochemica 2, 31 33
Steenwinckel, V. et al. (2007) J. Immunol., 178, 3244 3251
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Yu, Z. et al. (2007) Drug Metab. Dispos., 35, 981 986
Zelenay, S. et al. (2007) Int. Immunol., 19, 11 18
Solution-based Isolation
163