Quantitative RT-PCR using a PCR-generated competitive internal standard.

One approach to quantitate RNA is the use of a known number of competitive internal standard molecules that are reverse-transcribed and subsequently co-amplified under the same reaction conditions as the target sequence (3). This technique, called competitive reverse transcription polymerase chain reaction (cRT-PCR) has been applied to monitor the level of hepatitis C virus (HCV) RNA in patients’ serum samples (2,5). Quantitation of HCV RNA may have important implications for treatment strategies and clinical outcome (4). However, synthesis of competitive internal standard is labor-intensive, requiring the use of plasmid vectors and deletion techniques (2,5). We describe a simple method for quantitation of HCV RNA using an in vitro-synthesized, non-plasmid-generated HCV competitive internal standard. As shown in Figure 1, a 125-base single-stranded (ss)DNA fragment with T7 promoter that also contained sequences for HCV probe, sense and antisense primers was synthesized by a Model 391 PCR-Mate DNA Synthesizer (PE Applied Biosystems, Foster City, CA, USA). The ssDNA was then amplified for 40 cycles in a DNA Thermal Cycler 480 (Perkin-Elmer, Norwalk, CT, USA) in a 0.5-mL Eppendorf Safe-Lock Microcentrifuge Tube (Brinkmann Instruments, Westbury, NY, USA), overlaid with 50 μL of mineral oil with a set of primers: sense 5′-AATTTAATACGACTCACTATAGGGAACTG-3′ and antisense 5′-CGAGACCTCCCGGGGCACTC GCAAGCACCC-3′ to generate a doublestranded DNA. The PCR amplification mixture (final volume 50 μL) contained 5 μL 10× PCR buffer (100 mM TrisHCl, pH 8.3, 500 mM KCl, 15 mM MgCl2, 0.01% [wt/vol] gelatin), 0.5 μL AmpliTaq DNA Polymerase (5 U/μL), 8 μL 10 mM dNTP (both from Perkin-Elmer), 1 μL each of sense and antisense primer (50 pmol/μL), 33 μL distilled water and 30 pmol (2 μL) of the 125-base ssDNA. The amplification profile was as follows: denaturation at 94°C for 1 min, annealing at 55°C for 1 min and extension at 72°C for 1.5 min. Following the PCR, 5 μL of a 1:1000 dilution of the amplification product were subjected to two hours of in vitro transcription at 37°C using the Large Scale T7 Transcription Kit (Novagen, Madison, WI, USA) according to the manufacturer’s protocol. This was followed by digestion of the template DNA with 5 U of RNase-free DNase I (Novagen) at 37°C for 1 h. The synthesized RNA was extracted once with 100 μL of phenol/chloroform isoamyl alcohol (CIAA; 1:1; Amersham, Arlington Heights, IL, USA) and once with CIAA without phenol (Amersham). The extracted RNA was then precipitated in isopropanol, washed with 70% ethanol and resuspended in 100 μL of nuclease-free water. The RNA was further purified by passing through a Quick Spin G-50 Sephadex Column (Boehringer Mannheim, Indianapolis, IN, USA) according to the manufacturer’s instructions, and the amount of RNA was estimated by reading the absorbance at 260 nm in a UV spectrometer (UV/VIS Lambda 2; Perkin-Elmer). The optical density was translated to mg/mL, from which the number of amplicons was deduced. The presence of synthesized RNA and complete degradation of template DNA were confirmed by the lack of PCR product before, and its presence after, RT-PCR. Total nucleic acid was extracted from 100 μL of serum sample from an HCV-positive patient using the IsoQuick Nucleic Acid Extraction Kit (ORCA Research, Bothell, WA, USA) according to the manufacturer’s instructions. The extracted nucleic acid was then dissolved in 10 μL of nuclease-free water, and 2 μL of this were subjected to RT-PCR in the presence of increasing concentrations of competitor RNA molecules (102–108). Briefly, cRT was performed in a 0.2-mL MicroAmp tube (GeneAmp PCR System 9600; Perkin-Elmer) containing: 4 μL 25 mM MgCl2, 2 μL 10× PCR buffer II (100 mM Tris-HCl, 500 mM KCl, pH 8.3), 8 μL 10 mM dNTP, 1 μL (20 U/μL) RNase inhibitor, 1 μL (50 U/ μL) Maloney murine leukemia virus