ANALYTICAL BIOCHEMISTRY 75, 325-328 (1976)

Separation of High Molecular Weight Heterodisperse RNA and rRNA by Polyacrylamide Gels

A polyacrylamide gel containing a low percentage of bisacrylamide is shown to be applicable to separation of RNA molecules within the molecular weight range of 0.7 x IO6 to 15 x 106. the molecular weight of giant heterodisperse RNA. The exact relationship between mobility and molecular weight is reported.

When RNA was extracted from the epidermis cells of the fly Calliphora etythrocephala with the Pronase-phenol procedure (1) and electrophoresed in 2.2% gels of the composition given by Loening (2). a large portion of the labeled RNA remained in the two top slices of the gel. Similar results were obtained when the RNA was treated with formaldehyde (3) or a hot phenol procedure was used for RNA extraction (4). However, when the amount of bisacrylamide was lowered in the gel to a ratio of 1:48, thus increasing the pore size, all the radioactivity entered the gels. This modification allows the simultaneous resolution of 18 and 28s rRNA, 38s pre-rRNA, and heterodisperse RNA molecules in a molecular weight range up to 15 x 106.

METHODS

Modified gels of 2.2% polyacrylamide (10 x 0.6 cm) were prepared according to the method of Loening (2) with the exception that a ratio of total monomer to bisacrylamide of 48: 1 was used instead of the 20: 1 used by Loening. This modification was done in order to increase the pore size of the gel. Gels with a lower amount of bisacrylamide are very dif- ficult to handle. Composition of the electrophoresis buffer was 36 mM Tris, 34 mM NaH,PO,, 1 mM Na,-EDTA, 0.2% sodium dodecyl sulfate. Electrophoresis was carried out at 18°C and 6 mA/tube for 5 hr for Loening gels and for 4 hr for the modified gels. After electrophoresis the gel was scanned at 260 nm in a Gilford gel scanning apparatus. For measuring the radioactivity, the gel was sliced in 0.8-mm slices, placed in scintillation vials (two slices in each vial) containing 10 ml of toluene with 5 g of PPO and 40 ml of NCS (Nuclear Chicago) per liter, and hydrolyzed overnight at room temperature. The vials were counted in a Packard Tri-Carb liquid scintillation spectrometer at an efficiency of 35%. For molecular weight estimation Calliphora 38s pre-rRNA and 28 and 18s rRNAs were used as reference markers with molecular weights of 2.8 x 106, 1.45 x 106, and 0.71 x 106, respectively. The values for the mature rRNA were esti- mated for Chironomus rRNA (1) and adopted for Calliphora as the rRNAs of both species comigrate when they are coelectrophoresed. The value for the 38s precursor RNA was estimated in our laboratory. Calliphora

325 Copyright 0 1976 by Academic Ress. Inc. All rights of reproduction in any form reserved.

326 SHORT COMMUNICATIONS

FIG. 1. Electrophoretic separation of epidermis RNA extracted after a 60-min [3Hj- uridine pulse. (a), The ratio of total monomer to bisacrylamide was 20: 1, electrophoresis was for 5 hr. (b), The ratio of total monomer to bisacrylamide was 4&l, electrophoresis was for 4 hr. Sixty micrograms [3H]RNA was used for each experiment. Values of S refer to the absorbance peaks at 260 nm. The numbers represent the percentages of label measured for the various regions.

epidermis RNA extracted by the phenol-Pronase procedure (l), following a 60-min [3H]uridine pulse, was used for this study.

RESULTS

When Calliphora [3H]RNA was electrophoresed in 2.2% gels of the composition given by Loening (2), a large portion of the labeled RNA re- mained in the two top slices of the gel (Fig. la). However, by increasing the pore size of the gel by lowering the amount of bisacrylamide (see Methods), the distribution profile of radioactivity shown in Fig. lb was obtained. Practically all radioactivity has now entered the gel. In addition, the mobility of the defined RNA species increased, bringing the 18s rRNA to approximately the same position in the gel after a 4-hr run that it reached after approximately 5 hr in the gel with the higher percentage of bis- acrylamide. The two ribosomal RNA species in the larger-pore-size gel were clearly separated but were moving closer than in the smaller-pore- size gel (see Figs. la and b).

Profiles obtained with both types of gels had three peaks in common:

SHORT COMMUNICATIONS 327

those of the two species of mature 18 and 28s rRNA, labeled V in Figs. la and b, and the peak labeled IV, which represents the “38s” ribosomal precursor RNA. The 18 and 28s rRNA of Calfiphora had estimated molecular weights of 0.7 x lo6 and 1.45 x 106, respectively. Using these RNA species as standards and assuming a linear relation between mobility and log molecular weight (2), a molecular weight of approximately 2.8 x lo6 was estimated for the Calliphora 38s rRNA precursor regardless of which type of gel was used. This suggests that the relationship between mobility and log molecular weight is identical for the gels of the new composition and for those of Loening’s composition and is presum- ably linear in the range of ribosomal RNA sizes. In addition to the peaks of ribosomal and preribosomal RNA, the low-bisacrylamide gel profile showed one additional large peak of a mobility much slower than that of the 38s rRNA precursor (Fig. lb). If the relationship between molecular weight and mobility still holds for this molecular weight range, the RNA in this peak would have a molecular weight of about 15 x 106.

While our initial experiments strongly suggested that most of the material not entering the high-percentage bisacrylamide gels was contained in the slow-moving new peak of the low-percentage gels, they did not rule out the possibility that some of the ribosomal RNA or the hetero- disperse RNA of faster mobility was also retained in the high-percentage gels. Since this question is of particular importance for any attempt to quantitate the various species or size classes of RNA, the profiles were subdivided into five regions, as indicated in Figs. la and b. Region I con- tained all radioactivity moving more slowly than the 38s rRNA precursor: regions IV and V contained radioactivity attributed to the 38s pre-rRNA and the mature rRNA species, respectively; and regions II and III, heterodisperse RNA with mobility ranges of approximately 1.8-3.0 x lo6 and 0.6- 1.8 x IO6 daltons, respectively. As may be seen from Figs. la and b, the total counts recovered from the two gels were practically identical and, thus, the percentage of total counts calculated for each region. All radioactivity of RNA moving more slowly than 38s rRNA precursor in the high-percentage gels was contained in RNA moving more slowly than the rRNA precursor in the low-percentage gels. This means, however, that the material remaining on top of the high-percentage gels is identical with the material moving with a peak in the 15 x 106-dalton region of the low- percentage gels.

Therefore, we believe that this method will be useful for the isolation and characterization of high molecular weight heterodisperse RNA.

REFERENCES

1. Rubinstein, L., and Clever, U. (1971) Biochim. Biophys. Acta 246, 517-529. 2. Loening, U. E. (1%7) Biochem. J. 102, 251-257.

328 SHORT COMMUNICATIONS

3. Mayo, V. S., and DeKloet, S. R. (1971) Biochim. Biophys. Acta 247, 74-79.

4. Shaaya, E., and Clever, U. (1972) Biochim. Biophys. Acta 272, 373-381.

ELI SHAAYA

Laboratory of Insect Physiology

The Hebrew Universiry of Jerusalem

Terra Sancta, Israel Received February 6, 1976; accepted April 20, 1976