Solution-bas ed Iso lation

TriPure Iso lation Reagent

TriPure Isolation Reagent*

Clear, red so lutio n; ready -to-use
Cat. No. 1 667 157 (50 ml)
Cat. No. 1 667 165 (250 m l)

Principle During a one-step samp le homogen ization/lysis procedu re, the TriPure Iso-
la tion Reagent disrupts cells and dena tu res endogenous nucleases. After chlo-
roform is added to the extract, the mixture is centrifuged and separates into
three phases: a colorless aqueous (upper) phase, a white interpha se and a red
organic (lower) phase. The phases may then be separated and alcohol preci-
pita tion used to recover RNA (from the colorless aqueous phase), DNA and
protein (from the interphase and red organic phase).

4 Starting material 2 Cultured cells (research samples)

2 Fresh or frozen animal tissue (research samples)
2 Human leukocytes (research samples)
2 Ba cteria l cell suspensions
2 Yeast spheroplasts
2 Pla nt spheroplasts

Application 2 Prepa ration of total RNA, genomic DNA, and protein from a single bio-
logical sample
2 DNA-free total RNA may be used for Northern blots, in vitro translation,
RNa se protection assays, cDNA sy nthesis, or RT-P CR
2 RNA-free DNA may be used for P CR, restriction analy sis, Southern blots,
and cloning
2 Denatured protein may be used for SDS-PAGE and Western blots

Time required 2 Total time: approx. 2.5 h for RNA isolation

2 Hands-on time: approx. 25 min for RNA isolation

Results 2 Yields vary dependin g on starting material (See the table un der Part IV of
“How to use the reagent” in this article)
2 A2 60 /A2 8 0 of RNA = 1.6 – 2.0
2 A2 60 /A2 8 0 of DNA >1.7

Key 2 Saves time, beca use the RNA isolation procedure requires on ly 1 h
advantages
2 Easy to use, because the red dye in the reagent simplifies iden tifica tion of
differen t phases
2 Adapts eas ily to needs of specific laboratories, because the reagent can be
used with a wide va riety of starting samples
2 Simplifies isolatio n pro to cols, because a single reagen t can be used to isolate
DNA-free RNA, RNA-free DNA, a nd protein for a variety of applications
(see above)
2 Increases yield of intact RNA, because the reagent provides an immediate
chaotropic denaturin g environment that eliminates endogenous RNase
activity

* se e p a g e 2 33

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 So lutio n-based Isolatio n
TriPure Iso lation Reagent

How to use the reagent

I. Flow diagram For RNA isolation

(se e pa ge 1 5 4 ) 2 I sopropan ol
2 75% ethanol
2 Diethylpyrocarbonate (DE PC)-treated,
RNase-free water or DEP C-treated 0.5%
SDS

C o lor les s a q ueo us p h a se For DNA isolation

c o nta ining R N A
2 Absolute ethanol
W h it e inte rp h a se 2 75 % ethanol

R e d or g a nic p h a se
C ont ain D N A
a nd p ro te in
2 8 mM Na OH
2 0.1 M sodium citrate in 10 % ethanol
4
For protein isolation
2 I sopropan ol
II. Reagent contents
2 1 % sodium dodecyl su lfate (SDS)
TriP ure Isolation Reagent is a clear, red, mo-
nophasic solution of phen ol and guanidin e 2 0.3 M guanidine hydroch loride (GuHC l)
thiocyan ate, pH 4. It is ready to use as supp- in 95% ethanol
lied.
2 Absolute ethanol
For particular samples
III. Additional materials needed
For the extraction and phase separation 2 H omogen ization apparatus (for tissue an d
protocol certain cells only )
2 Sterile, disposable poly propylene tubes 2 Red Blood Cell Lysis Buffer, Cat. No.
tha t can withstand 12,000 x g in th e 1 814 389 (for white blood cells only)
presence of TriPure Isolation Reagen t an d
2 Glycogen (for processing <10 mg tissue)
chloroform
2 C hloroform (free of all additives su ch as
isoamyl alcohol)

IV. Average nucleic acid yield from various sources

Sample RNA yield DNA yield

Tissue:
Liver 6 – 10 µg/mg tissue 3 – 4 µg/mg tissue
Spleen 6 – 10 µg/mg tissue not determined
Kidney 3 – 4 µg/ mg tissue 3 – 4 µg/mg tissue
Skeletal muscle or brain 1.0 – 1.5 µg/mg tissue 2 – 3 µg/mg tissue
Placenta 1 – 4 µg/ mg tissue 2 – 3 µg/mg tissue

Cultured cells:
Epithelial cells 8 – 15 µg/10 6 cells not determined
Fibroblasts 5 – 7 µg/10 6 cells not determined
Hu ma n, mouse, or rat cells not determined 5 – 7 µg/10 6 cells

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 Solution-bas ed Iso lation
TriPure Iso lation Reagent

V. Protocol for preparing RNA, DNA, and protein from animal tissue
(based o n the m ethod of Chom czynski, 1993)
Note: For a detailed, step-by-step procedure and fo r tips on handling different ty pes of sam ple, see the pack age insert supplied w ith the
reagent.

Extraction
Add 1 ml TriP ure per 50 – 100 mg tissue
Homogenize sample in tissue homogenizer

Incubate 5 min at 15 to 25° C to dissociate nucleoprotein complexes

4 Add chloroform (0.2 ml per 1 ml TriPure)

Shake vigorously 15 s; I ncubate 2 – 15 min at 15 to 25° C

Centrifuge 12,000 x g, 15 min at 2 to 8°C

3 Phases: Aqueous (contain ing RNA ) colorless

In terphase (containing DNA) white
Organ ic (con taining protein) red

Isolation of RNA Isolation of DNA/protein

Transfer aqueous phase to from rema ining interphase a nd organic phase
new tube

Precipitate with P recipitate with etha nol (EtOH)

isopropanol (0.3 ml 100% EtOH per 1 ml TriPu re)
(0.5 ml per 1 ml TriPu re) M ix by inversion
M ix by inversion Incu ba te 2 – 3 min at 15 to 25° C
Incubate 5 – 10 min at
15 to 25°C

Centrifuge 12,000 x g, Cen trifu ge 2000 x g, 5 min at 2 to 8° C

10 min at 2 to 8° C Sepa rate pellet and phenol/EtOH supernatant
Discard suppernatan t

Wash pellet 1 x with 75% Isolation of DNA Isolation of protein

EtOH (1 ml EtOH per Pellet P henol/EtOH supernatant
1 ml TriPure)

C entrifuge 7500 x g, Wa sh pellet in 10% EtOH /0.1 M sodium Precipitate with isopropa nol
5 min at 2 to 8°C citrate (1 ml per 1 ml TriPure) (1.5 ml per 1 ml TriPure)
Discard supern atant Incubate 30 min at 15 to 25°C with Mix by in version
occasional mixing In cubate 10 min at 15 to 25°C

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 So lutio n-based Isolatio n
TriPure Iso lation Reagent

Air dry pellet C entrifuge 2000 x g, Cen trifuge 12,000 x g,

Resu spend in RNase-free 5 min at 2 to 8°C 10 min at 2 to 8°C
water or DEPC -trea ted Discard supern atant Discard supernatant
0.5% SDS
Incubate 10 – 15 min at
55° – 60°C to resuspend

Store RNA at –15 Repeat wash step 2 x Resuspen d pellet in

to –25°C 0.3 M Gu HCl in 95% EtOH
(2 ml per 1 ml TriPure)
Incubate pellet in wash 20 min at 15 to 25°C

Wash pellet in 75% EtOH

(1.5 – 2.0 ml per 1 ml TriPure)
Cen trifuge 7500 x g, 5 min at 2 to 8° C
Discard supernatant
4
Incubate 10 – 20 min at 15 to 25°C with
occasional mixing

Centrifuge 2000 x g, 5 min at 2 to 8° C Repeat wash step 2 x

Discard supern atant

Briefly dry pellet Wash protein pellet in

5 – 10 min under vacuum (or air dry) 2 ml 100% EtOH
Dissolve pellet in 8 mM NaOH Vortex
Incubate 20 min at 15 to 25°C

Adjust pH to 7.0 – 8.0 with HE PES Centrifuge at 7500 x g, 5 min a t 2 to 8°C

Buffer an d adjust to 1 mM EDTA Discard supernatant

Store DNA a t 2 to 8°C Dry pellet un der vacuum 5 – 10 min

(or air dry)
Add 1% SDS a nd in cubate at 50°C to
dissolve pellet
Sediment insoluble material 10,000 x g,
10 min at 2 to 8°C

Transfer su pernatan t to new tu be

Store protein at –15 to –25°C

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 Solution-bas ed Iso lation
TriPure Iso lation Reagent
V. Troubleshooting the TriPure Isolation protocol
During If you get... Then, the cause may be...
RNA isolation Low RNA yield Incomplete homogen ization or lysis of
sa mples
Incomplete solubilization of the final
RNA pellet
A2 6 0 /A28 0 ra tio Insufficien t TriP ure used for sample
<1.65 homogenization
4 After h omogen ization, samples were
not stored for 5 min at 15 to 25°C
Contamination of aqueous pha se with
phenol phase
Incomplete solubilization of the final
RNA pellet
RNA Tissues were not immedia tely processed
degradation or frozen after removal from animal
Samp les u sed for isolation procedure
were stored at –20°C instead of –70°C
Cells were dispersed by trypsin digestion
Aqueous solutions or tubes were n ot
RNase-free
DNA Insufficien t TriP ure used for sample
contamination homogenization
Sta rting samples contained organic
solvents (E tO H, DM SO) or strong bu ffers;
or had an a lkaline pH
156
And you should...
- Use power homogenizer to maximize
sa mple yields.
- Do not let RNA pellet dry completely, as a
dry pellet will be much less soluble.
- Increase incubation time to 30 min at 55°C
to solubilize RNA.
- Add a sufficient volume of TriP ure Isolati-
on Reagen t, according to pa ckage insert
instru ctions.
- Store at 15 to 25°C for 5 min.
- Carefully remove the upper aqueous phase
for subsequent RNA iolation, making sure
to avoid the interpha se/organic phase.
- Increase incubation time to 30 min at 55°C
to solubilize RNA.
- Use fresh tissue or tissue that has been di-
rectly frozen in liquid nitrogen and stored
at –70°C prior to RNA isolation .
- Add TriPure I solation Reagent directly to
cells attached to culture dish or flask, ac-
cording to package insert instructions.
- Use sterile disposable pla sticware and pi-
pettes/tips reserved for RNA work only.
- Take a ppropriate precau tions to ensure
RNase-free en vironment.
- Add a sufficient volume of TriP ure Isolati-
on Reagen t, according to pa ckage insert
instru ctions.
- Carefully remove the upper aqueous phase
for su bsequent RNA isolation, making
sure to avoid the interphase/organic pha se.
 So lutio n-based Isolatio n
TriPure Iso lation Reagent

V. Troubleshooting the TriPure Isolation protoc ol, c ontinued

During If you get... Then, the cause may be... And you should...

DNA isolation L ow DNA y ield In complete homogenization or lysis of - Use power homogenizer to maximize
samples sample yields.

In complete solubilization of the final - Do not let DNA pellet dry completely, as a
DNA pellet dry pellet will be much less soluble.

A26 0 /A2 80 ratio In complete removal of phenol from the - I ncorpora te an a ddition al sodium citra te/
<1.7 DNA prepara tion (during ethanol/ ethan ol wash step.
sodium citrate wash)

DNA Tissues were not immediately processed - Use fresh tissue or tissue that has been
degrada tion or frozen a fter remova l from an imal
Samples used for isolation procedure
directly frozen in liquid nitrogen and
stored at –70° C prior to DNA isolation. 4
were stored at –20° C instead of –70°C

Samples were homogenized with a high - Avoid using power homogenizer. Use
speed homogenizer ha nd-held homogenizer to minimize shea-
rin g of high molecular weight DNA.

RNA Too much aqueous phase remain ed with - C arefully remove a ll of the upper aqueous
con ta mination the interphase a nd organic phase pha se prior to isolation of DNA.

In adequate wa sh of DNA pellet with - After addin g 1 ml sodium citrate/ethanol

10% EtOH/0.1 M sodium citrate for each 1 ml TriPure Isola tion Reagen t
(required in the initial homogenization
process) incubate the samp le, with occa-
sional mixin g, for 30 min at 15 to 25° C.

Protein L ow protein In complete homogenization or lysis of - Use power homogenizer to maximize

isolation y ield samples sample yields.

In complete solubilization of the final - I ncubate sample a t 50°C to completely

protein pellet solubilize th e protein.

P rotein Tissues were not immediately processed - Use fresh tissue or tissue that has been
degrada tion or frozen a fter remova l from an imal directly frozen in liquid nitrogen and
stored at –70° C prior to protein isolation.

Deformed Protein pellet not washed sufficiently - I ncorpora te an additiona l wash step.
ba nds in PA GE
a naly sis

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 Solution-bas ed Iso lation
TriPure Iso lation Reagent

Typical result with the reagent

1 2 3 4 5 6 7 8 9 10 11 12 13

4
/
Figu re 52. N orth ern blo t with to tal RN A isolate d by
the TriPure Isola tion Rea gent. T o ta l R N A w a s iso la te d
( b y th e T r iP ur e p ro to c o l) fr o m th e fo llo w in g sa m p le s:
6 7
1 .5 x 10 c ells of a h um an le uke m ia c ell line , 5. 0 x 1 0
7
h um a n w h ite b lo o d ce lls, 1. 7 x 1 0 b uff y c oa t ce lls fro m
h um a n b lo o d, and 5 00 m g ra t tis sue . Th e iso la te d RN A
sa m p les w er e se p a ra te d ele ctr o p h o re tica lly o n a g e l,
tr a nsfe rr ed t o a ny lo n m e m b ra ne , a nd h yb r idize d w ith a
1 k b , dig o xig e nin-la b ele d g lyce ra lde h yde 3-p h o sp h a te
de h ydr og ena se (G 3 P D H ) p r o b e . T h e b lo t w a s incub a te d
o ve rnig h t w ith D IG /G e nius S y ste m r ea g e nts fo r c h e mi-
lum ine sc e nt de te ctio n, th e n e xp o se d to X-r ay film fo r 5
m in. Th e G 3 P D H p ro b e re c o g nize s a 1. 3 5 kb m RN A , a s
sh o w n in:
Lan e 1: R N A ladde r
Lan es 2 – 4 : RN A fr o m h uma n le uke m ia c e ll line
Lanes 5 – 7: RN A f ro m h um an w h it e b lo o d ce ll p e llet
Lan es 8 – 1 0: RN A fro m h um a n b lo o d b uffy co a t; and
Lan es 11 – 13: R N A fro m r at live r t iss ue
T h e am o unt o f t ot al RN A ap p lied t o t h e o r ig ina l g el w a s
e ith e r 5 µ g (lane s 2 , 5, 8 , 1 1) , 1 µ g ( lane s 3, 6 , 9 , 12 ) ,
o r 0. 2 5 µ g (la nes 4, 7 , 1 0 , 1 3 ).
Result: Th is da ta cle ar ly dem o nstr a te s th at h ig h q ua lity ,
inta ct R N A is s ucc es sfully iso la ted fr o m a va rie ty o f sta r-
ting m a te rials us ing t h e T r iP ur e Is ola tio n Re ag e nt.

References
Bakker, O. (1998) Biochemica 1, 22-23;
Biochemica Information (1998) 102, 30-31
Chomczynski, P. (1993) BioTechniques 15, 532–537.
Garry D. et al. (1996) Nature 395, 905-908
Morris, T. et al. (1996)
J. Clin. Microbiology 2933-2936
Schummer, B. et al. (1998)
Technical Tip Biochemica 2, 31-33
Yin Hu et al. (1997)
Molecular Pharmacology 51, 370-376

158