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Nucleic Acids Research, Vol. 19, No. 12 3251 Polyinosinlc acid as a carrier in the microscale purification of total RNA Steven G.Winslow* and Pierre A.Henkart Experimental Immunology Branch, National Cancer Institute, Building 10, Room 4B17, Bethesda, MD 20892, USA Received April 1, 1991; Accepted May 6, 1991 ABSTRACT Three different RNA carriers were compared for use in microscale RNA isolation and subsequent cDNA synthesis and amplification via the polymerase chain reaction. E.coli rRNA alone gave considerable cDNA synthesis which under standard carrier conditions overwhelmed cDNA synthesis from lymphocyte mRNA. Yeast tRNA caused inhibition of mRNA primed cONA synthesis, giving low levels of cDNA synthesis when used without cellular RNA. In contrast, commercially available poly I alone did not prime detectable cDNA synthesis nor did it inhibit such synthesis primed by cellular mRNA. When RNA preparations were made using these three carriers and decreasing numbers of starting lymphocytes, poly I allowed the detection of cDNA from two orders of magnitude fewer lymphocytes than the other carriers. Thus poly I was found to be a superior carrier molecule for microscale RNA preparations suitable for reverse transcription and subsequent amplification using the polymerase chain reaction. INTRODUCTION Recent reports have shown that PCR amplification of cDNA can be utilized to analyze mRNA from very limited numbers of cells (1 —3). In scaling down standard protocols for RNA purification, it is particularly difficult to obtain quantitative precipitation of small amounts of RNA. A standard approach to this problem involves addition of an irrelevant nucleic acid species which acts as a carrier to enhance the quantitative recovery of precipitated products. In surveying the literature for a suitable carrier for microscale RNA preparations, we found that some studies had used E.coli rRNA (1,2). In preparing RNA from small numbers of mouse lymphocytes with this carrier followed by reverse transcription and polymerase chain reaction (RTPCR), we found either no recovery of PCR product or the amplification of a number of unexpected bands. We decided to systematically compare the efficacy of E.coli rRNA, yeast tRNA and the commercially available synthetic polyribonucleotide polyinosinic acid (poly I) as carriers and to determine their effects on the synthesis at the cDNA level. Our results, presented here, show • To whom correspondence should be addressed that poly I is an excellent carrier molecule which shows little tendency to interfere with cDNA synthesis of cellular RNA and subsequent PCR amplification. MATERIALS AND METHODS RNA preparation. Cloned murine cytotoxic T lymphocytes recognizing the class I MHC molecule H-2 Kb were the source of cellular RNA in these experiments. The method for isolation of RNA was a microscale version of Chomczynski and Sacchi (4) with the addition of carrier molecules. All steps were carried out in 1.5ml siliconized microfuge tubes. A pellet containing the indicated number of cells was solubilized in 250/il of solution D (4M GITC, 25mM sodium citrate pH 7.0, 0.5% sarcosyl, 1 % 2-mercaptoethanol (vol/vol), and 0.1% antifoam A) along with 3jtl of carrier RNA stock solution (lOmg/ml) which was added prior to shearing the DNA. Carriers used were: (1) E.coli rRNA (Sigma); (2) yeast tRNA (BRL); or (3) polyinosinic acid (Pharmacia # 27-4330). The final RNA pellet was dried under vacuum (Speed Vac Concentrator, Savant Instruments) and then resuspended in DEPC treated H2O to an appropriate volume (5/tl for microscale preparations). cDNA synthesis. For direct analysis of cDNA synthesis, a 15/x.l cDNA synthesis reaction mixture was prepared containing: 1^1 of dATP, dGTP, and dTTP lOmM stocks (Perkin Elmer Cetus); 5^1 32P dCTP (3000^Ci/mmol); 2/tl I O X A M V - R T buffer (500mM Tris-HCl at pH 8.3, 500mM KC1, lOOmM MgCl2, lOmM DTT, lOmM EDTA, 100/tg/ml BSA); 1^1 lOmM spermidine; 2y\ of 50/tM random hexamer (Pharmacia) in TE, pH 8.0; 1/tl RNAsin (27U//tl Boehringer Mannheim Biochemicals); 1/xl AMV-RT (25U//tl Boehringer Mannheim Biochemicals that was diluted to 10U//tl in 10% glycerol, lOmM potassium phosphate pH 7.4, 0.2% Triton X-100, and 2mM dithiothreitol prior to addition to reaction)(5). RNA samples were heated to 90°C for 5 minutes and cooled on ice. The cDNA synthesis reaction mixture was mixed with the RNA sample (in 5/il DEPC-treated H2O) giving a total reaction volume of 20/xl. The sample was incubated at 43° C for 15 minutes followed by a chase of l/il of each lOmM dNTP solution. The reaction was then incubated for another 30 minutes at 43°C. Upon completion, 3252 Nucleic Acids Research, Vol. 19, No. 12 the reaction was heated at 95 °C for 5 minutes to inactivate the reverse transcriptase. For reactions in which the cDNA was amplified by PCR, 4/tl of DEPC-H2O and \y\ of cold dCTP (lOmM) was used in place of the 32P dCTP and the incubation was carried out for 45 min with no chase. Electrophoretic Analysis ofcDNA. The RNA was hydrolyzed in the cDNA reactions by the addition of an equal volume of 0.3N NaOH/30mM EDTA and 5 minutes of boiling. The reaction was neutralized with 1/4 the original volume (approximately 5/*l) of 1M Tris pH 8.0. The unincorporated 32P dCTP was removed with Elutip-D (Schleicher & Schuell) as per manufacturers instructions. Cleaned samples were precipitated with 2 volumes EtOH for 1 hour at -20°C. Samples were centrifiiged at 10,000g for 30 minutes and then dried under vacuum. The labelled cDNA was resuspended in 25/d DEPC treated H2O. The samples were analyzed by alkaline agarose electrophoresis (6) using 10/il from each sample preparation. The gel was fixed in 7% TCA for 45 minutes with shaking and blotted dry overnight. Autoradiography was done the next day for 1 hour at room temperature and then photographed. Polymerase Chain Reaction. A PCR reaction mixture was designed to amplify cDNA from j32-microglobulin, which is well expressed on the lymphocytes used. This mixture consisted of: 5/tl each of 20/tM sense (5'-GTATGCTATCCAGAAAACCCCTC-3') and antisense (5'-CATGTCTCGATCCCAGTAGACGG-3') oligonucleotides in TE, 65jtl sterile dH2O, 20/tl of master mix, and 5/d of cDNA reaction. The master mix consisted of: 10 x PCR reaction buffer, 1.25mM final concentration of each dNTP, and 2.5U AmpliTaq per reaction (Perkin Elmer Cetus). All reactions were overlaid with 50/xl of mineral oil. The PCR was preformed for 40 cycles in a single block thermocycler (Ericomp) with the following profile: (1) 20 seconds at 94°C, (2) 1 minute at 55 °C and (3) 2 minutes at 72 °C. Samples of 20^1 were analyzed on 2% agarose ethidium bromide gels run at 200V for 45 minutes to 1 hour. seen that the use of the E.coli rRNA and yeast tRNA carriers distort the quantity and quality of cDNA compared to RNA prepared without carriers (Lane 1) or RNA prepared with the poly I carrier (Lane 4). The amount of label incorporated into cDNA was clearly increased in the presence of E.coli rRNA carrier, and decreased in the presence of yeast tRNA. We infer that the tRNA inhibits cDNA synthesis primed by the cellular RNA. The residual product made in the presence of tRNA is much smaller in size than the control in lane 1 and is the approximate size predicted for a full length tRNA cDNA transcript. In contrast to these carrier molecules, when an equal amount of poly I was added to the reaction Qane 4), the cDNA products appear similar in size and intensity to the reaction in the absence of carrier. Figure 2 shows the results obtained by alkaline agarose gel analysis of products of the reverse transcriptase reaction carried out with the carrier molecules alone, with lymphocyte RNA as a positive control (lane 1). A large amount of cDNA product was seen with the E.coli rRNA (lane 2) and and a small amount with yeast tRNA (lane 3); both patterns appear similar to those when lymphocyte RNA was present (Fig. 1). This implies that the bulk of cDNA synthesis in the presence of these carrier molecules was primed from the carrier and not from the cellular RNA. In contrast, the poly I alone fails to prime detectable cDNA synthesis (lane 4). 1 2345 -5kb l| -2 -1 -0.5 RESULTS AND DISCUSSION Figure 1 shows a comparison of the cDNA products of the reverse transcriptase reaction on mouse lymphocyte RNA in the presence of different carrier RNA preparations. It can be clearly 12 3 4 5 Figure 2. Autoradiograph of cDNA synthesis reactions from the carriers alone. Lane 1, approximately 0.5^g TIM-CTL total RNA as a reference. Lane 2, 0.5 Hg E.coli rRNA; lane 3, 0.5 fig yeast tRNA; lane 4, 0.5 /tg poly I. Lane 5 is a control in which no RNA was added to the cDNA synthesis reaction. A 123456 B123456 C123456 -5kb -2 -1 -0.5 Figure 1. Autoradiograph of cDNA synthesis reactions on an alkaline agarose gel. Each of four parallel cDNA reactions were carried out as described in the presence of 32 P dCTP with approximately 0.5/ig total TTM-CTL RNA and 0.5 /tg of the various carriers. The products of these reactions were electrophoresed in denaturing gels and autoradiography used to determine the size and intensity of the cDNA products. The carriers used were as follows: Lane 1, none; lane 2, E.coli rRNA; lane 3, yeast tRNA, and lane 4, polyinosink acid. Lane 5 was a control with no RNA added to the cDNA reaction. Figure 3. Products of RTPCR for lymphocyte beta-2-microglobulin mRNA. Ethidium bromide stained 2% agarose gels are shown. In each panel lanes I - 6 represent one fourth of the PCR product derived from decreasing numbers of starting cells for independent RNA preparations. The predicted length of PCR product from /J2-microglobulin mRNA from the oligos used is 290 bp. The DNA molecular weight standard in the left lane of each panel is a Hae III digest of phi-X 174, with the fifth largest band being 310 bp. Shown are ten-fold serial dilutions of cells from 100,000 (lane 1) to 1 (lane 6) in the initial RNA preparation. The earners were: Panel A, poly I; panel B, E.coli rRNA; panel C, yeast tRNA. NucUic Acids Research, Vol. 19, No. 12 3253 Figure 3 shows the results of RTPCR for beta-2-microglobulin mRNA prepared with different carriers using decreasing cell numbers. It can be seen that poly I is a clearly superior carrier, giving detectable amplified cDNA products with 100 fold fewer cells than the rRNA or tRNA carriers. In other experiments, microscale lymphocyte RNA preparations with the rRNA carrier followed by RTPCR, gave unexpected DNA bands as well as a poor yield of the expected product when compared to larger scale RNA preparations. In contrast, use of poly I carrier in microscale preparations has consistently given results which were in line with those obtained in macroscale preparations without carrier. These results are consistent with the experiments shown in Figs. 1 and 2. They suggest that poly I is a superior carrier for such small scale RNA preparations. The use of this synthetic RNA precludes the presence of any contaminating mRNA or genomic DNA which could be present in preparations of cellderived RNA and which could be responsible for the unexpected products of PCR amplification of the cDNA. Furthermore, cost comparisons indicate that poly I is the least expensive of the carriers tested on a weight basis. REFERENCES 1. Rappolee,D.A., Wang,A., Mark.D., and Werb.Z. (1989) J. Cellular Biochem. 39:1-11. 2. Brenner, C.A., Tam.A.W., Nelson,P.A., Engelman.E.G., Suzuki,N., Fry,K.E., and LarrickJ.W. (1989) Biotechiuques 7:1096-2002. 3. Akowitz.A., and Manuelidis.L. (1989) Gene 81:295-306. 4. Chomczynski,P. and Sacctu,N. (1987) Analytical Biochemistry 162:156-159. 5. Berger.S.L., and Kimmel.A.R. (1987) Meth. in Enzymol. 152:316-325 6. ManiatisX, Fritsch.E.F, and Sambrook.J. (1982) Molecular Cloning: A Laboartory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY.