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Llll|Llll Technical Tips Downloaded from genome.cshlp.org on October 5, 2021 - Published by Cold Spring Harbor Laboratory Press llll|llll Technical Tips The reverse transcriptase-polymerase reverse transcriptase, the multiple Single-step chain reaction (RT-PCR) provides an ef- rounds of amplification catalyzed by Elimination of fective method for detecting very small DNA polymerase are equally effective amounts of a specific mRNA in a small at amplifying either the cDNA or con- Contaminating sample of total RNA.O,2) Unfortunate- taminating genomic DNA. Even mi- DNA Prior to ly, for purposes of detecting RNA by nuscule amounts of contaminating this procedure, after the initial step of DNA (<1%) can produce a false- Reverse converting RNA into cDNA using positive amplification signal in an RT- Transcriptase PCR Donald D. Dilworth and John R. McCarrey Department of Genetics, Southwest Foundation for Biomedical Research, San Antonio, Texas 78228 FIGURE 1 Effects of pretreatment with nuclease on production of amplification signals by RT-PCR. Five hundred nanograms of total RNA from mouse liver supplemented with 0, 1, 5, or 10% genomic DNA was subjected to RT-PCR using primers determined from the mouse 13- actin cDNA sequence, (14) and reverse transcriptase and AmpliTaq DNA polymerase (Perkin- Elmer Cetus). Primers used were: (upstream) 5'-GCGGACTGTTACTGAGCTGCGT-3' and (downstream) 5 ' -GAAGCAATGCTGTCACCTTCCC-3 ', which delineated a 453-bp amplifica- tion product from either a cDNA or genomic DNA template, since this sequence apparently does not span an intron in genomic DNA. In each case a comparison of amplification with (+) or without (-) the addition of reverse transcriptase is shown. (A) RT-PCR with no prior nuclease treatment. Note amplification signal in both +RT and -RT lanes, with increasing in- tensity in -RT lane coincident with increasing levels of contaminating DNA. The faint signal in the 0% DNA/-RT lane indicates endogenous DNA contamination in the RNA preparation, which represents <1% of the total nucleic acid. (B) RT-PCR following treament with DNase I. Note persistent signal in +RT lanes, but absence of signal in -RT lanes. (C) RT-PCR following treatment with both DNase I and RNases A and T1. Note the absence of amplification signal in both the +RT and -RT lanes. Restriction endonuclease cleavage of the amplification pro- duct with Rsal was used to confirm amplification of the f~-actin template in each case (data not shown). A 123-bp DNA ladder (BRL) was run in each of the outside lanes to confirm molecular size of the amplification product. 1:279-2829 by Cold Spring Harbor Laboratory Press ISSN 1054-9803/92 $3.00 PCR Methods and Applications 279 Downloaded from genome.cshlp.org on October 5, 2021 - Published by Cold Spring Harbor Laboratory Press Technical PCR reaction (Fig. 1). One method to tions in 20 ~tl = S mM MgC12, lx PCR no-RT (-RT) lanes. Note that con- distinguish amplification signals tem- buffer (SO mM KC1, 10 mM Tris-HCl, pH taminating DNA present at levels of plated by genomic DNA versus mRNA- 8.3), 1 mM each dNTPs, 20 units of less than 1% is sufficient to produce a derived cDNA is to use primers that RNase inhibitor, 2.5 ~tM random hexa- noticable amplification signal. Figure 1 span an exon-exon junction in the mers (note: the DNase treatment did B shows that treatment with DNase I cDNA, such that the presence of an in- not seem to affect these short, single- effectively eliminates the DNA signal tron in the genomic DNA template will stranded primers)]. Contaminating (no band in -RT lanes), but leaves the yield a larger amplification product DNA was removed by adding 0.4 ~tl of RNA signal intact (signal in +RT lanes). than that emanating from the intron- 0.25 ,g/~tl RNase-free DNase I (U.S. That the signals in the +RT lanes in less cDNA template. (3) However the Biochemical~ and incubating at 37~ Figure 1B were indeed produced by necessary sequence information to de- for 30 rain. To test the potential extent RNA templates was confirmed by treat- sign such primers (i.e., location of in- of elimination of genomic DNA by this ing with DNase plus RNase, which trons) is not always known for a partic- method, some RNA samples were eliminated the signals from both the ular gene, and some functional genes spiked with varying amounts of geno- +RT and -RT lanes, as shown in Figure lack introns, as do all processed pseu- mic DNA isolated from mouse liver as 1C. These results demonstrate that the dogenes. Thus, it is often desirable to described. (4) The reaction was stopped DNase I treatment we describe can be able to eliminate contaminating by heating to 95~ for 5 min, then eliminate up to 10% of contaminating genomic DNA directly from samples of cooled to room temperature. One mi- DNA from a 500-ng sample of total RNA prior to RT-PCR. croliter of 50 U/,l reverse transcriptase RNA, which represents a much higher DNA can be eliminated from RNA was then added and the RT-PCR proce- level of contamination than normally samples by treatment with RNase-free dure followed according to manufac- observed. In fact, we found that this DNase I as a separate step in the turer's instructions, including one treatment can eliminate up to 100% purification of total RNA.(4-6) However round of reverse transcription (42~ DNA (500 ng) (data not shown), in- RT-PCR is often used to analyze very 15 min; 99~ 5 min; 5~ 5 min) in a dicating that it is sufficient to remove small samples of total RNA, and at- total volume of 20 ~tl, followed by ad- any amount of contaminating DNA tempts to recover such small amounts dition of 80 ~tl of PCR master mix con- from a standard 500-ng RNA sample. of RNA following a separate DNase di- taining Taq polymerase and primers The absence of a signal in any of gestion step can result in the loss of all specific for mouse cytoskeletal ~-actin the no-RT lanes in Figure 1B indicates nucleic acid, including RNA. Recovery (final concentrations in 100 ~tl -- 2 mM that under these conditions the can be augmented by addition of car- MgC12, lx PCR buffer, 2.5 units of AmpliTaq DNA polymerase used does rier RNA, but this introduces another AmpliTaq DNA polymerase, and 125 not show RT activity. We also tested potential source of amplification ng each of upstream and downstream the native Taq polymerase for RT ac- artifact. We have developed a one-step specific primers), and 35 cycles of PCR tivity under our RT-PCR conditions, procedure for the removal of con- (each cycle - 94~ 1 min; 54~ 1 and again observed no signal in the taminating genomic DNA from RNA min; 72~ 1 rain, with a 5 sec/cycle -RT lanes (Fig. 2). Finally we tested samples that can be carried out in the extension of the 72~ step). Fifteen mi- both AmpliTaq and native Taq, along same tube as the subsequent RT-PCR croliters of reaction product was then with two other thermostable DNA reaction, without addition of carrier electrophoresed through 1.5% agarose polymerases, Vent (New England RNA. We show here that this proce- and visualized by staining with ethid- Biolabs) and Pfu (Stratagene), for ap- dure can effectively remove even very ium bromide. "No-RT" controls were parent RT activity under standard DNA large amounts of contaminating run as above except that water was PCR conditions by testing each on genomic DNA, with no appreciable substituted for reverse transcriptase. DNA versus RNA templates (Fig. 3). We loss of RNA. Treatment of some samples with both observed no evidence for any RT ac- RNase and DNase was performed as an tivity in the AmpliTaq, native Taq, or Pfu polymerases under the conditions METHODS additional control by adding 0.1 ~tg RT-PCR was performed using the RNase A and 5 units of RNase T1 described. The Vent polymerase did GeneAmp RNA PCR kit (Perkin-Elmer (GIBCO-BRL) to the DNase step and produce a faint amplification product Cetus) according to manufacturer's in- reducing the water to keep the total from DNase I-treated RNA, suggesting structions, modified to facilitate treat- volume at 19 ~tl. the possibility of very minor RT ac- ment of the sample with DNase I. The tivity associated with this enzyme un- DNase I treatment was carried out in RESULTS AND DISCUSSION der these conditions. the same reaction mix as the reverse The presence or absence of an RT-PCR The occurrence of RT activity in transcriptase step, prior to addition of amplification signal emanating from one thermostable DNA polymerase, the reverse transcriptase. Five hundred contaminating genomic DNA can be Thermus thermophilus (rTth) DNA nanograms of total RNA isolated from detected by running a "no-RT" (no polymerase (Perkin-Elmer Cetus), in mouse liver as described (7) in 2 ~tl of reverse transcriptase added) control. the presence of manganese ion is well diethyl pyrocarbonate (DEPC)-treated Figure 1A shows the presence of con- documented (8) and undisputed. How- water was mixed with 16.6 ~tl of reverse taminating DNA in samples of total ever, reports of RT activity associated transcriptase master mix excluding RNA that were not treated with DNase with the Taq polymerases are more var- reverse transcriptase [final concentra- I, as indicated by positive signals in the iable. Myers and Gelfand (8) reported 280 PCR Methods and Applications Downloaded from genome.cshlp.org on October 5, 2021 - Published by Cold Spring Harbor Laboratory Press llllll! Technical Tips FIGURE 2 Analysis of AmpliTaq and native Taq polymerases for reverse transcriptase ac- tivity during RT-PCR.
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