Download Polyinosinlc acid as a carrier in the microscale purification of total RNA

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
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l|
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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
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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.