Single-step elimination of contaminating DNA prior to reverse transcriptase PCR.

Abstract

The reverse transcriptase-polymerase chain reaction (RT-PCR) provides an effective m e t h o d for detecting very small a m o u n t s of a specific mRNA in a small sample of total RNA.O,2) Unfortunately, for purposes of detecting RNA by this procedure, after the initial step of converting RNA i n t o cDNA using reverse transcriptase, the multiple rounds of amplification catalyzed by DNA polymerase are equally effective at a m p l i f y i n g either the cDNA or cont a m i n a t i n g g e n o m i c DNA. Even minuscule a m o u n t s of c o n t a m i n a t i n g DNA (<1%) can produce a falsepositive amplification signal in an RT-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 (PerkinElmer Cetus). Primers used were: (upstream) 5'-GCGGACTGTTACTGAGCTGCGT-3' and (downstream) 5 ' -GAAGCAATGCTGTCACCTTCCC-3 ', which delineated a 453-bp amplification 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. PCR Methods and Applications 279Cold Spring Harbor Laboratory Press on June 7, 2019 -Published by genome.cshlp.org Downloaded from Technical PCR reaction (Fig. 1). One method to distinguish amplification signals templated by genomic DNA versus mRNAderived cDNA is to use primers that span an exon-exon junction in the cDNA, such that the presence of an intron in the genomic DNA template will yield a larger amplification product than that emanating from the intronless cDNA template. (3) However the necessary sequence information to design such primers (i.e., location of introns) is not always known for a particular gene, and some functional genes lack introns, as do all processed pseudogenes. Thus, it is often desirable to be able to eliminate contaminating genomic DNA directly from samples of RNA prior to RT-PCR.DNA can be eliminated from RNA samples by treatment with RNase-free DNase I as a separate step in the purification of total RNA. (4-6) However RT-PCR is often used to analyze very small samples of total RNA, and attempts to recover such small amounts of RNA following a separate DNase digestion step can result in the loss of all nucleic acid, including RNA. Recovery can be augmented by addition of carrier RNA, but this introduces another potential source of amplification artifact. We have developed a one-step procedure for the removal of contaminating genomic DNA from RNA samples that can be carried out in the same tube as the subsequent RT-PCR reaction, without addition of carrier RNA. We show here that this procedure can effectively remove even very large amounts of contaminating genomic DNA, with no appreciable loss of RNA. METHODSRT-PCR was perf...

Putative markers for the detection of breast carcinoma cells in blood

“…Reverse transcription of RNA and PCR amplification of cDNA was carried out using RNA-PCR Kit (GeneAmp RNA-PCR kit, Perkin-Elmer Cetus, Norwalk, CT, USA) after treatment of samples with RNAase-free DNAase I (Dilworth and McCarrey, 1992). The PCR reaction was carried out in a Perkin-Elmer Cetus 480 DNA thermal cycler; the PCR conditions for each set of primers are given below.…”

Reduced Myocardial Sarcoplasmic Reticulum Ca 2+ -ATPase mRNA Expression and Biphasic Force-Frequency Relations in Patients With Hypertrophic Cardiomyopathy

“…The DNAse I treatment was carried out in the same reaction mixture as the RT before addition of reverse transcriptase to remove contaminating genomic DNA, as described by Dilworth and McCarrey. 17 The mRNA expression of Ca 2ϩ -handling proteins was analyzed by a fluorogenic 5Ј-nuclease PCR assay using an ABI PRISM 7700 sequence detector (Perkin-Elmer). 18 The cDNA products were amplified with an initial denaturation at 95°C for 10 minutes and 50 cycles of PCR, with each cycle consisting of denaturation at 95°C for 15 seconds and annealing and extension at 60°C for 60 seconds (TaqMan PCR core kit, Perkin-Elmer).…”

“…cDNA was synthesized from 300 ng of total RNA and human heart total RNA (Clontech) at 42°C for 50 minutes with oligo(dT) [12][13][14][15][16][17][18] and Moloney murine leukemia virus RT (SuperScriptII, Gibco BRL). The DNAse I treatment was carried out in the same reaction mixture as the RT before addition of reverse transcriptase to remove contaminating genomic DNA, as described by Dilworth and McCarrey.…”

Evidence for the Presence of 5S rRNA in Mammalian Mitochondria

“…Total RNA from 293T cells that had been transfected transiently with p5SSE or pSV2neo was subjected to RT-PCR with either primers 5S-F and 5Sm-R (5Ј-AAAGCCTACAGCACCCG-3Ј) or primers 5S-C (5Ј-GGCCTGGTTAGTACTTGG-3Ј) and 5Sm-R, followed by SmaI digestion, labeling, and electrophoresis through a nondenaturing polyacrylamide gel. Organellar RNA was treated with DNase I as described (Dilworth and McCarrey, 1992) before RT-PCR. …”