CA1105402A - Isolation of undegraded rna - Google Patents

Abstract

ABSTRACT OF THE DISCLOSURE
RNAse is effectively inhibited during isolation of messenger RNA (mRNA) from cells or tissues by homogenizing a cell preparation in guanadinium thiocyanate buffered to low pH and containing mercaptoethanol. The combination of the added chemicals renders the inactivation of RNase essentially irreversible, allowing the mRNA to be isolated intact.

Description

54~2 ll Isolation of intact messenger RNA (mRNA) from cells or tissues is !¦ technically extremely difficult due to the presence of the enzyme ribo-I' nuclease (RNase) which catalyzes ~he hydrolysis of a single phosphodiesterj bond in the ribonucleotide sequence. This en~yme is ubiquitous, axtremely ¦ stable and highly active. It is found on the skin, survives ordinary glassware washing techniqùes and sometimes con~aminates stocks of organic chemicals. The hydrolysis of a single phosphodiester bond within the mRN~
chain cannot be tolerated~ when the mRNA is isolated for cloning purposes, ,, since that would destroy the sequence continuity necessary to preserve ,i the genetic lnformation.
The difficul~ie~ are especially acute in dealing ~ith extracts of l! pancreas cells 7 since the pancreas is a source of digesti~e enzymes and ¦¦ therefore, is rich in RNase. HGwever, the problem of RNase contamination i is present in all tissues. The method disclosed herein to eliminate R~ase activity is applicable to all tissues. The exceptional effectiveness of the method is demonstrated in the present invention by the successful isolation of intact mRNA from isolated islet cells of the pancreas.

Prior Art RNase is a name applied collectively to a group of similar en~ymes 0 1 found to some extent in virtually all biological materials. Pancreatic RNase is a fairly typical example, and its structure is ~uite ~ell known.
The enzyme i5 a relatively low molecular weight protein possessing inter-molecular disulfide bonds. Catalytic activity is depen~ent upon a certain . . , . ~

a54q12 folded configuration of the amino acid chain, termed the native con-figuration. Denaturation is the name given to any process which leads to unfolding or rearrangement of the amino acid chain such that the nati~e, catalytically active configuration is lost.
Denaturation of RNase is relatively diff~cult to achieve. The inter--molecular disulfide bonds maintain the amino acid chain in a configuration approximating the native configuration. If the disulfide bonds are left intact, renaturation occurs rapidly, upon removal o~ denaturant. Even I when the disulfide bonds are broken, the enzyme is capable of spontaneously I recovering the native configuration under appropriate conditions, although li, the process is much slower.
¦¦ The denaturation of RNase by the use of chaotropic ions has been il reported. For a general discussion of chaotropes and denaturation, see I¦ Jencks, W.P., Catalysis in Chemistry and Enzymology, Chapt. 7 (1969). In ~I particular, the denaturation of RNase by guanidinium thlocyanate, a salt having both a chaotropic anion and a chaotropic cation, has been demon- !
strated by VonHippel, P.H. and Wong, K-Y, Science 145, 577 (1964).
Numerous strategies have been employed in the prior art to isolate j undegraded RNA from biological sources. The success of such procedures jj has been defined relative to the nature and purpose of the RNA isolation ¦l and to the amount of degradation which could be tolerated. One strategy employed in the prior art is the use of an adsorbant, such as bentonite, to remove RNase from the cell extract. Another strategy has been the ' complete deproteinization of the cell e~tract. Phenol i9 commonly used ii for this purpose. Guanidinium hydrochloride has also been employed as a li deproteinizing agent for the preparation of nucleic acids, and its use has been reviewed by Cox, R.A., Methods in Enzymology (L. Grossman and K. Moldave, eds.) vol. 12B, 120 (196~). Although such procedures may ¦I be employed to isolate high molecular weight RNA, they are not adequate !i for the isolation of mRNA for cloning purposes, where even the hydrolysis of a single phosphodiester bond would render the entire molecule useless ~ 354U2 for the purpose of transferring an intact gel~etic sequence to a micro-organism. The difficulties are exacerbated when the isolation is attempted from a tissue high in RNase content, such as pancreas. Rat pancreas contains as much as 200JUg of RNase per gram of tissue and even higher amounts are found to occur in other mammalian pancreases.

SUMMARY OF THE INVENTION
RNase is effectively inhibited during isolation ~rom intact cells by homogenizing a cell preparation in guanidinium thiocyanate buffered to low 1~ pH and containing mercaptoethanol. In combination with the denaturing , action of guanidinium thiocyanate, mercaptoethanol further reduces RNase Il acti~ity by disrupting its intermolecular disulfide bonds. The combination i of mercaptoethanol with a potent denaturing agent such as guanidinium 1, j thiocyanate, pursuant to this invention, tends to enhance the effectiveness Il of mercaptoethanol by rendering the inactivation of RNase essentially ,` irreversible. The pH may be varied in the range Df 5.0 - 8Ø
., , " DETAILED DESCRI~TION OF THE INVENTION -The present invention employs in combination a chaotropic anion, a chaotropic cation and a disulfide bond-breaking agent, during cell I disruption and during all operations required to separate RNA essentially ~I free from protein. The effectiveness of the combined action of the fore-going agents has been demonstrated by their use in the isolation of essentially undegraded mRNA in good yield from isolated islets of Langerhans of rat pancreas.
jl Choice of suitable chaotropic ions is based upon their solubility in li aqueous media and upon availability. Suitable chaotropic cations include li guanidinium, carbamoylguanidinium, guanylguanidinium, lithium and the like.
¦l Suitable chaotropic anions include iodide, perchlorate, thiocyanate, j diiodosalicylate and the like. The relatiYe effectiveness of salts formed 1l by combining such cations and anions will be determined in par~ by their l i , I ~, I! .

solubility. For example, lithium diiodosalicylate is a more potent denaturan~---than guanidinium thiocyanate, but it has a solubility of only about 0.lM and is also relatively expen-sive. Guanidinium thiocyanate provides the preferred cation-anion con~ination, because it is readily available and is highly soluble in aqueous media, up to about 5M.
Thiol compounds, such as ~-mercaptoethanol, are ~nown to break intramolecular disulfide bonds in proteins by a thiol-disulfide interchange reaction. Many suitable thiol compounds are known to be effective, including besides ~-mercaptoethanol, dithiothreitol, cysteine, propanol di-mercaptan and the like. Aqueous solubility is a necessary property, since the thiol compound must be present in large excess over the intramolecular disulfides, in order to drive the interchange reaction essentially to completion. ~-mercapto-ethanol is preferred because of its ready availability at reasonable price.
For the purpose of inhibiting RNase during extrac-tion of RNA from cells or tissues, the effectiveness of a given chaotropic salt is directly related to its concentra-tion. The preferred concentration is therefore the highest concentration that can be employed, as a practical matter.
The success of the present invention in preserving mRNA intact during extraction is thought to depend upon the rapidity with which the RNase is denatured, in addition to the extent of denaturation. This is thought to explain, for example, the superio~ity of guanidinium thiocyanate over the hydrochloride, despite the fact that the hydrochloride is only slightly less potent as a denaturing agent. The efectiveness of a denaturant is defined as the threshold concentration needed to achieve complete denaturation o a protein. On the other hand, the rate of denaturation of many proteins is ~ ~5~

dependent on the denaturant concentration, relative to the threshold, raised to from -the 5th to the 10th power. See Tanford, C.A., Ad~. Prot. Chem. 23, 121 (1968). Qualitatively this relationship suggests that a denaturant only slightly more potent than guanidinium hydrochloride can denature a protein many times more rapidly a~ the same concentration.
The relationship between the kinetics of RNase , 54~;~

denaturation and the preservation of mRNA during its extraction from cells I is not thought to have been recognized or exploited, prior to the present ¦ invention. The foregoing analysis, if correct, suggests that the preferred ¦ denaturant will be one having a low threshold denaturing concentration ¦ combined with a high aqueous solubility. For this reason, guanidinium thiocyanate is preferred over lithium diiodosalicylate even thou~h the ¦ letter is a more potent denaturant~ because the solubility of guanidinium I thiocyanate is much greater, hence it can be used at a concentration ¦I permitting more rapid RNase inactivation. The foregoing analysis also I explains why guanidinium thiocyanate is preferred over the comparably soluble hydrochloride salt, since the former is a somewhat more potent l denaturant , '.
The use of a disulfide bond breaking agent in combination with a ~ denaturant potentiates and enhances the effectiveness of the latter by 1I permitting the RNase molecule to become completely unfolded. The thiol compound is thought to enhance the forward rate of the denaturation process by preventing the rapid renaturation which can occur when the intramolecular disulfide bonds are left intact. Furthermore, any conta~inating RNase ~ remaining in the mRNA preparation will remain substantially inactive, even ~i in the absence of the denaturant and thiol. Disulfide bond breaking agents having thiol groups will be effective to some extent at any concentration, although generally speaking, a large excess of thiol groups to intra-molecular disulfide bonds is preferred to drive the interchange reaction ~` in the direction of intramolecular disulfide cleavage. On the other l hand, many thiol compounds are malodorous and unpleasant to work with j in high concentration, so that a practical upper concentration limit ¦ exists. Using ~-mercaptoethanol, concentrations in the range from .05M
to l.OM have been found effective, and 0.2M is considered optimal, for I¦ the isolation of undegraded in RNA from rat pancreas.
1I The pH of the medium during extraction of mRNA from cells may be l anywhere in the rang`e of pH 5.0 - 8Ø

l l l . .

I ~5-Example Islet cells of rat pancreas were prepared according to ~opending U S application 805,023, incorporated herein by reference.
~ ~ Islet cells pooled from 2~0 rats were homogenized in 4 M guanidinium thiocyanate containing ~ mercaptoethanol buffered to pH 5.0 at 4C.
The homogenate was layered over 1.2 ml, 5.7 M CsCl containing lOO mM EDTA
and centrifuged for 18 hours at 37,000 rpm in the SW 50.1 rotor of a Beckman Ultracentrifuge at 15C (Beckman Instrument Company, Fullerton, I California). RNA traveled to the bottom of the tube.
lll Polyadenylated RNA was isolated by chromatography of the total RNA
preparation on oligo(dT)-cellulose according to the procedure of Aviv, H., and Leder, P., supra.
Avian myeloblastosis virus reverse transcriptase, provided by D.J.
~i Beard, Life Science Inc., St. Petersburg, Florida, was used to transcribe i total polyadenylated RNA from rat islets of Langerhans into cDNA. The ,~ reactions were carried out in 50 mM Tris-HCl, pH 8.3 9 mM MgC12, 30 mM NaCl, 20 mM beta-mercaptoethanol, 1 m~I each of 3 nonradioactive deoxyribonucleoside triphosphates, 250JuM of the fourth deoxynucleoside triphosphate labeled with d<_32p, specific activity 50-200 curies per mole, 20~ug/ml oligo-dT12_1g 1i from Collaborative Research9 Waltham, Massachusetts, 100Jug/ml polyadenylatedRNA and 200 units/ml reverse transcriptase. The mi~ture was incubated at !~ 45C for fifteen minutes. After addition of EDTA-Na2 to 25 mM, the solution ,i was extracted with an equal volume of water-saturated phcnol, followed by I' chromatography of the aqueous phase on a Sephadex G-100 column, 0O3 cm in ~ diameter by 10 cm in height, in 10 mM Tris-HCl, pH 9.0, 100 mM NaC1, 2 mM
I EDTA. Nucleic acid eluted in the void volume was precipltated with ethanol ¦l after addition of ammonium acetate, pH 6.0, to 0.25M. The precipitate was collected by centrifugation, the pellet was dissolved in 50,ul of freshly '! prepared 0.1M NaOH and incubated at 70C for 20 minutes to hydrolyze the ii RNA. The mixture was neutralized by the addition of lM sodium acetate, pH

~ 1/ Tridom, trademark, Fluke AG Chemische Fabrik, Buchs, Switzerland.
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¦1 4.5, and the 32P-cDNA product was precipitated with ethanol and redissolved in water. Aliquots of single stranged cDNA were analyzed on native polyacrylamide gels by the method of Dingman, C.~., and Peacock, A.C., 1 Biochemistry 7, ~59 (1968). The gels were dried and the 32p DNA detected 1 by autoradiography using Kodak No-Screen NS-2T ~ilm. The cDNA was 1 heterodisperse, as judged by the electrophoresis pattern. It contained at ! least one prominent cDNA species of about 450 nucleotides, as judged by ! I comparison with known standards.
I¦ The cDNA of about 450 nucleotides in length was cloned by transfer '', to and replication in a bacterial strain, reisolated and subjected to sequence analysis. It was found to comprise all but the first five nucleotides of the coding sequence for rat proinsulin I, as well as a portion of the 3'-untranslated region. Bearing in mind that length hetero-,' geneity is introduced during reverse transcription of the isolated mRNA
" template, it was concluded that the mRNA had been isolated substantially undegraded from the rat pancreas.

GENERAL CONCLUDING REMARKS
The ability to isolate undegraded mRNA according the present invention I represents a substantial improvement over prior art procedures. The mRN~
l~ has been isolated, as described herein, from a tissue well known to contain large amounts of RNase. The disclosed method and reagent composition used to inhibit RNase under these conditions is suitable for use in the `` isolation of RNA from other sources having comparable or lesser amounts ., , ~, of RNase, and is also considered suitable for RNA isolation from sources ., , i containing even greater amounts of RNase.
,1 While the invention has been described in connection with specific embodiments thereof, it will be understood that it is capable of further modifications and this application is intended to cover any variations, ,~ uses, or adaptations of the invention following, in general, the principles , of the invention and including such departures from the present disclosure :

2/ Trademark, Eastman Kodak Corporation, Rochester, New York.

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~54~2 I as come within known or customary practice within the art to which the ¦ invention pertains or which would be readily apparent to those skilled ¦l in said art. With that ~mderstanding, the invention is not to be limited except to t~e e~tent required by the appended tlaims.

Claims (8)

The embodiments of the invention in which an exclusive property or privilege is claimed are defined as follows:

1. A method of preparing a cell extract essentially free of RNase activity, comprising:
homogenizing a population of cells at controlled pH in the combined presence of a protein denaturing agent and a disulfide bond breaking agent.

2. The method of claim 1 wherein the protein denaturing agent is guanidinium thiocyanate and the disulfide bond breaking agent is mercap-toethanol.

3. An RNase inhibitor composition comprising a chaotropic cation, a chaotropic anion and a disulfide bond breaking agent.

4. A composition according to claim 3 wherein the chaotrepic cation is guanidinium and the chaotropic anion is thiocyanate.

5. A composition according to claim 3 wherein the disulfide bond disrupting agent is .beta.-mercaptoethanol.

6. A composition according to claim 3 comprising 4 M guanidinium thiocyanate and 0.2 M .beta.-mercaptoethanol.

7. A method of isolating substantially undegraded RNA from cells comprising:
extracting RNA from the cells in the combined presence of a protein denaturing agent and a disulfide bond breaking agent, and fractionating the cell extract to separate RNA from protein, whereby substantially undegraded RNA is isolated.

8. The method of claim 7 wherein the protein denaturing agent is guanidinium thiocyanate and the disulfide bond breaking agent is .beta.-mercaptoethanol.