Use of Elevated Reverse Transcription Reaction Temperatures in RT-PCR

The use of reverse transcriptase coupled with the polymerase chain reaction (RT-PCR) has been proven to be a valuable tool in the synthesis and isolation of cDNA clones as well as the quantification of rare messenger RNA species. While a number of different approaches can be used to permit gene-specific cDNA synthesis and amplification, a recurring problem is one of high background signal (i.e., mispriming). A primary contributor to this problem may be that the RT reaction is traditionally performed at the optimal temperature for reverse transcriptase, 42°C. At this temperature and routine salt concentrations (50 to 100 mM), the initial primer annealing step is normally far below the melting temperature (Tm) of the 3′ primer. This can produce illegitimate (unwanted) hybridization and the subsequent amplification of unexpected PCR products. We report here that a potential solution to this problem is to increase the stringency of the RT step by using elevated reaction temperatures. In fact, the use of elevated RT reaction temperatures (50°–55°C) has been utilized to overcome problems of mRNA secondary structure (1,3,5). As a model system, a synthetic RNA for the dopamine D2 receptor (4) was amplified in an RT-PCR using sequence-specific primers. Briefly, 10 pg (121 fmol) of in vitro-synthesized D2 RNA were converted to a first-strand cDNA using a 3′-specific primer (5′-TCTGCGGCTCATCGTCTTAAG-3′; Tm = 64°C at 2°C per A/T and 4°C per G/C). The RT reaction was performed in the presence of 1 μM primer and 5 units of avian myeloblastosis virus (AMV)-reverse transcriptase (Seikagaku America, Ijamsville, MD, USA). The final concentrations of the RT buffer components were: 10 mM Tris-HCl (pH 8.3 at 4°C), 50 mM KCl, 2.5 mM MgCl2, 0.005% Tween 20, 0.005% Nonidet P-40, 10 μg/mL gelatin, 500 units/mL RNasin (Promega, Madison, WI, USA) and 1.25 mM of each deoxyribonucleoside triphosphate in a final volume of 20 μL. The cDNA synthesis reactions were performed at a number of temperatures between 42°C and 60°C for 60 min. A portion of the RT reaction (2.5 μL) was then amplified by PCR. The PCR step was conducted in a total volume of 25 μL containing the following components (exclusive of those constituents contributed by the RT reaction): 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 0.1% Triton X-100, 2.5 mM MgCl2, 100 μCi/mL [α-32P]dCTP, 50 units/mL Taq DNA Polymerase (Perkin-Elmer, Norwalk, CT, USA), 1 μM of the 3′-primer described above and 1 μM of a gene-specific 5′-primer (5′-GCAGTCGAGCTTTCAGAGCC-3′, Tm = 64°C). The amplification was performed through 20 PCR cycles (94°C for 1.5 min, 65°C for 2 min and 72°C for 2 min) following initiation using a “hot-start” addition of the polymerase. The “hot start” entailed 5 min at 94°C during which the polymerase was added through the mineral oil layer. The products were resolved on a nondenaturing 10% polyacrylamide gel (2). The gel was then dried and the products visualized by autoradiography. A Betascope 603 Blot Analyzer (Betagen, Waltham, MA, USA) was used to directly quantify the radiolabeled products.