Hybridization of DNA to RNA in methylmercuric hydroxide agarose gels

Journal of Biochemical and Biophysical Methods, 9 (1984) 215-220 Elsevier

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BBM00415

Hybridization of DNA to RNA in methylmercuric hydroxide agarose gels Alex Elbrecht and C a t h e r i n e B. L a z i e r * Biochemistry Department, Dalhousie University, Halifax, Nova Scotia, Canada B3H 4H7

(Received 28 November1983) (Accepted 13 February 1984)

Summa~ We describe a method for hybridization of cDNA probes to RNA directly in agarose gels which provides a practical alternative to methods involving transfer of the RNA out of the gel. Total cellular RNA is subjected to electrophoresis in agarose gels containing methylmercuric hydroxide as the denaturing agent. After removal of the methylmercuric hydroxide, the gel is dried and 32p-labeled DNA probes are hybridized to the immobilized RNA. This method is more economical in time and expense than methods involving transfer of the RNA out of the gel, while maintaining a level of sensitivity comparable to other procedures. Key words: D N A - R N A hybridization; agarose gel electrophoresis~ methylmercuric hydroxide.

Introduction The Southern [1] and so-called 'northern' [2,3] hybridization techniques are commonly used procedures in molecular biology. Both methods involve electrophoresis of DNA or RNA samples in agarose gels, followed by transfer and binding of the nucleic acid to nitrocellulose or diazobenzyloxymethyl-paper. Identification of specific nucleic acid sequences is then accomplished by hybridizing labeled probes to the nucleic acids bound to the solid supports. Alternative methods have been described for hybridizing labeled probes to DNA or RNA directly in the agarose gel used to resolve the nucleic acid samples. Shinnick et al. [4] developed a procedure for hybridizing RNA to DNA in agarose gels at about the same time as Southern [1] developed his transfer method. More recently, Stiegler et al. [5] and Tsao et al. [6] have described a method for hybridizing DNA * To whom correspondence should be addressed. Abbreviations: apo-ll, apoprotein-II of very low density lipoprotein; cDNA, complementary DNA. 0165-022X/84/$03.00 © 1984 Elsevier Science Publishers B.V.

216 probes to RNA in agarose gels after electrophoresis using urea and glyoxal respectively as the denaturing agents. Accurate molecular weights for RNA molecules can be obtained from their mobilities during electrophoresis only if the secondary structures formed by the single-stranded molecules are completely denatured. Methylmercuric hydroxide (CHaHgOH) is a reversible denaturing agent that completely destroys all secondary structure in RNA and DNA molecules [7]. This agent is useful for samples where glyoxal or urea is not effective in completely denaturing the RNA. When CH3HgOH is used in agarose gels to denature the nucleic acid, a linear curve is obtained for a plot of mobility versus log10 molecular weight [7]. We have developed a procedure for hybridizing 32P-labeled DNA probes to RNA directly in agarose gels after electrophoresis in the presence of CH3HgOH as denaturing agent. After electrophoresis, it is not necessary to transfer the RNA out of the gel onto nitrocellulose or diazobenzyloxymethyl-paper. The CH3HgOH can be removed from the gel by washing with buffer containing sulfhydryl compounds. The gel can then be dried and used for hybridization. We have used this procedure to identify specific mRNA species in total cellular RNA from embryonic chick liver, and find that the sensitivity of this method is comparable to others involving transfer of the RNA out of the gel.

Materials and Methods

Methylmercuric hydroxide was obtained as a 1 M aqueous solution from Alfa Products (Danvers, Mass., U.S.A.). CH3HgOH is extremely toxic and should always be handled in the fume hood [7]. We took the added precaution of using a No. 8707 mercury vapor respirator (3M Co., St. Paul, Minn., U.S.A.). Nitrocellulose membrane filters (0.45 /~m) were from Schleicher and Schuell (Keene, N.H., U.S.A.). [~-32p]dATP (5000 Ci/mmol) was synthesized by Dr. J. Hofman of Dalhousie University. Total cellular RNA was isolated from pooled embryonic chick livers by a modification of the method of Cox [8] as described by Protter et al. [9]. Poly(U) Sephadex G-10 was used as recommended by the manufacturer (Bethesda Research Laboratories Inc., Rockville, Md., U.S.A.) to prepare RNA fractions enriched for poly(A) + -RNA. Two recombinant plasmids designated CSA 44 and apo-II 60 were gifts of Dr. D.L. Williams (State University of New York at Stony Brook, N.Y., U.S.A.). The recombinant plasmid CSA 44 consists of a 2200 base pair cDNA of chicken serum albumin mRNA inserted into the Pst 1 site of the vector pBR322 [10]. Apo-II 60 contains an 800 base pair cDNA fragment of very low density lipoprotein apoprotein-II mRNA inserted into the Pst 1 site of pBR322 [9]. A third recombinant plasmid designated A26, a gift of Dr. R.G. Deeley (Queen's University, Kingston, Ont., Canada), contains a 520 base pair cDNA of chicken serum albumin mRNA inserted at the Pst 1 site of PBR322, and has been described previously [11]. Plasmids were labeled with [a-32p]dATP by nick translation [11], or by using T 4

217 DNA polymerase as directed by the supplier (Bethesda Research Laboratories Inc.), and were used at specific activities of approximately 1 × 10 6 cpm//~g. Agarose gels containing CH3HgOH were prepared according to the method of Bailey and Davidson [7] using a horizontal slab gel apparatus. RNA was transferred out of the gel onto nitrocellulose filters and hybridization was carried out as described by Thomas [3]. For hybridization in the agarose gel, total cellular RNA was subjected to electrophoresis, in a fume hood, through an agarose gel containing 10 mM CH3HgOH. After electrophoresis, the gel was washed in a baking dish twice for 20 min periods with 500 ml wash buffer: 10 mM dithiothreitol/100 mM flmercaptoethanol/1 × SSC (150 mM sodium chloride/15 mM sodium citrate, pH 6.5) and once for 20 min with 1 × SSC, pH 7.0. CH3HgOH reacts readily with sulfhydryl compounds [7] and these washes should be sufficient both to inactivate and to remove the CH3HgOH from the gel. The gel was then dried onto 3MM chromatography paper (Whatman Ltd., U.K.) using a commercial gel drying apparatus (BioRad Laboratories, Mississauga, Canada). During this procedure the upper surface of the gel was covered with Handi-wrap. The dried gel was removed from the chromatography paper by dipping it briefly in distilled water. The gel can be cut to a desired size before or after drying. The gel was then prehybridized, hybridized and washed as described by Thomas [3], except that the gel was washed four times for 10 min each at room temperature and twice for 30 min each at 50°C with gentle shaking. Prehybridization and hybridization were carried out in polyethylene zip-lock seal bags at 42°C. After washing the gel was blotted dry, wrapped in Handi-wrap, and subjected to autoradiography with Kodak XAR-5 film and Dupont Cronex Lighting Plus intensifying screens at - 7 0 ° C . After autoradiography, RNA in the dried gel can be visualized by staining the gel with ethidium bromide.

Results and Discussion

We have used both a northern transfer method and the in-gel hybridization technique to identify specific hepatic mRNA molecules at different stages of chick embryo development. For both procedures, mRNAs were identified by hybridization with 32p-labeled recombinant plasmids containing sequences complementary to either apo-II mRNA (Fig. 1A) or chicken serum albumin mRNA (Fig. 1B, C). Each panel consists of paired autoradiographs from in-gel hybridizations and northern blots. In panel A, the samples resolved by electrophoresis in lanes 1 and 2 were total hepatic cellular RNA from control and estrogen-treated chick embryos, respectively. Samples resolved in lanes 3 and 4 were hepatic poly(A)+-RNA from control and estrogen-treated chick embryos. With both methods the apo-II cDNA probes hybridize to only one band in the lanes containing RNA samples from the livers of estrogen-treated embryos. This is the expected result since expression of this particular gene, the apo-II gene, depends on the presence of the inducer estrogen [13]. In the absence of estrogen, the apo-II gene is not transcribed and therefore no

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1:2

3~4

Fig. 1. In-gel hybridization of mRNA with apo-I1 and chicken serum albumin cDNA probes. (A) Detection of apo-II mRNA by in-gel hybridization and after northern transfer. For the autoradiograph of lanes 1 and 2, total cellular RNA was resolved on a 2% (w/v) agarose gel containing 10 mM CH3HgOH, as described in Materials and Methods section, and hybridized, while still in the gel, to a 240 bp Eco R1 cDNA fragment from plasmid apo-ll 60 labeled with 32p using T4 DNA polymerase. Lane 1 was loaded with 55 ~g of total cellular RNA from livers of control 11 day old embryos and lane 2 with 50 ttg of total cellular RNA from livers of estrogen-treated 11-day-old embryos. For the autoradiograph of lanes 3 and 4, poly(A)+-RNA was resolved on a 1.5% (w/v) agarose gel containing 5 mM CH3HgOH and transferred to nitrocellulose paper for hybridization with nick translated apo-II 60 plasmid. Lane 3 was loaded with 4 gg of poly(A)+-RNA from livers of control 15 day old embryos and lane 4 with 4/~g of poly(A)+-RNA from livers of estrogen-treated 15 day old embryos. (B) In-gel detection of the chicken serum albumin mRNA. For lane 1, total cellular RNA (100 ~g from control 16-day-old embryos) was resolved on a 1.5% (w/v) agarose gel containing 10 mM CH3HgOH and hybridized with nick-translated CSA A26 probe while still in the gel. The gel was not prehybridized and the time of exposure to X-ray film was decreased because of the intense background. For the autoradiograph of lane 2, poly(A)+-RNA (4 /,tg from 15-day-old estrogen-treated embryos) was resolved on a 1.5% (w/v) agarose gel containing 5 mM CH3HgOH and subsequently transferred to a nitrocellulose filter, and hybridized with nick-translated probe CSA 44. (C) Direct comparison of in-gel hybridization and hybridization after northern transfer. Hepatic total cellular RNA (20/~g, lanes 1 and 4; 50 p,g, lanes 2 and 3) from estrogen-treated 15-day-old embryos was resolved on a 1.5% (w/v) agarose gel containing 10 mM CH3HgOH, After electrophoresis, half of the gel (lanes 1 and 2) was processed for in-gel hybridization and the RNA in lanes 3 and 4 was transferred for 20 h to a nitrocellulose filter. Prehybridization, hybridization with the same nick-translated CSA 44 probe, washing and autoradiography were identical for the dried gel and the nitrocellulose filter. s p e c i f i c h y b r i d i z a t i o n s h o u l d b e seen. B o t h m e t h o d s e x h i b i t a l o w b a c k g r o u n d a n d i n n e i t h e r o f t h e c o n t r o l s ( l a n e s 1 a n d 3) w a s s p e c i f i c h y b r i d i z a t i o n d e t e c t a b l e . U s i n g a D N A - e x c e s s s o l u t i o n h y b r i d i z a t i o n a s s a y , we h a v e d e t e r m i n e d t h e number of molecules of apo-II mRNA present in chick embryo hepatocytes ( m a n u s c r i p t i n p r e p a r a t i o n ) . I n t h e livers o f e s t r o g e n - t r e a t e d c h i c k e m b r y o s t h e r e a r e a p p r o x i m a t e l y 3 0 0 0 a p o - I I m R N A m o l e c u l e s a t d a y 11 o f e m b r y o n i c d e v e l o p m e n t , which represent approximately 0.01% of total cellular RNA, and approximately 6500 m o l e c u l e s a t d a y 15, w h i c h r e p r e s e n t a p p r o x i m a t e l y 0.02% o f t o t a l c e l l u l a r R N A .

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Thus, with probes of relatively low specific activity, as employed here (approximately 1 × 106 c p m / ~ g DNA), and using 50~tg of total RNA, specific RNA molecules that make up approximately 0.01% of total RNA can be detected (Fig. 1A, lane 2). Using probes with specific activities of 108 cpm//~g, and based on a fl-globin mRNA content of 0.1% in total erythrocyte mRNA, Thomas [3] was able to detect 50 pg of specific mRNA in 0.05/~g of total RNA. We are able to detect 5 ng of specific apo-II mRNA in 50/~g of total RNA using probes with specific activities of the order 10 6 cpm/~g. Presumably a 100-fold increase in the specific activity of the probes would result in a sensitivity comparable to that obtained by Thomas [3] with the transfer method. A direct comparison of the in-gel hybridization procedure with a northern blot is presented in panel C. Total hepatic cellular R N A from estrogen-treated 15-day-old embryos (20/~g, lanes 1 and 4; 50 ~g, lanes 2 and 3) was resolved in an agarose gel containing 10 mM CH3HgOH. After electrophoresis, half of the gel was processed for in-gel hybridization (lanes 1 and 2) and the RNA in the other half was transferred to a nitrocellulose filter (lanes 3 and 4). Both were hybridized with the same amount of the same probe (CSA 44), washed, and exposed to X-ray film for the same period of time. This shows that the sensitivities of both procedures are approximately equivalent. We find that for this procedure, as for others [3], the best ~'esults are obtained when the hybridization buffer contains formamide. At the hybridization temperature used, high concentrations of formamide favor the formation of R N A : D N A hybrids over D N A : D N A duplexes [14]. Prehybridization of the dried gel with heterologous DNA, to block non-specific binding of the probe D N A to the gel, reduces the background. The effect of prehybridization can be seen in Fig. lB. Lane 1 is taken from an autoradiograph of a gel where the RNA was not transferred out of the gel and the dried gel was not prehybridized. Lane 2 is from an autoradiograph of RNA transferred to a nitrocellulose filter. The background in lane 1 is more intense than that in lane 2, and also greater than the background in panels A and C. Thus high background levels of nonspecific DNA binding can be reduced by prehybridizing the gel as in Fig. 1 (A, C). One possible drawback of this procedure is the vacuum drying method. Small polynucleotides can be lost from the gel during the drying process. For example, a considerable portion of small DNA fragments less than 250 base pairs in length was lost from a 3% ( w / v ) agarose gel (results not shown). Of course, the loss of any particular nucleic acid molecule is inversely proportional to its size and the concentration of the agarose gel. A different drying procedure that avoids suction [6,15] may be better suited for in-gel hybridizations involving small RNAs.

Simplified description of the method and its applications The method we have described combines agarose gel electrophoresis, using CH3HgOH as the denaturing agent, with an in-gel hybridization procedure. CH3HgOH is an effective denaturing agent,

220 especially for larger RNA molecules or RNA molecules possessing extensive secondary structures [7,16]. Since the transfer step has been eliminated, this method is less time consuming and there is no need for expensive nitrocellulose or other RNA-binding substrates. Elimination of the transfer step also means that it is not necessary to consider the efficiency of transfer of RNA out of the gel, or the efficiency and extent of binding of RNA to the nitrocellulose. The sensitivity of the in-gel hybridization procedure is similar to other methods involving transfer of the R N A out of the gel.

Acknowledgements W e t h a n k D r . R . E . P e a r l m a n f o r m a k i n g a v a i l a b l e his u n p u b l i s h e d m a n u s c r i p t o n a n i n - g e l D N A : D N A h y b r i d i z a t i o n p r o c e d u r e a n d Dr. R. S i n g e r f o r h e l p w i t h t h e p r e p a r a t i o n of this paper. G r a n t s u p p o r t was f r o m the Medical R e s e a r c h Council of Canada (MT4880).

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