Preparing polyA-containing RNA internal standards for multiplex competitive RT-PCR.

PCR-based strategies such as competitive RT-PCR or semiquantitative RTPCR are commonly used for quantitating low-abundance mRNA transcripts (4,7–9). Competitive RT-PCR relies on amplifying the same target with a known amount of a competitor/reference sequence and comparing the relative amounts of the two PCR products. To improve the sensitivity and accuracy of these methods, often expensive and cumbersome techniques such as HPLC or the use of radioactivity have been used for PCR product quantitation (1,2, 6,10). Ideally, a good competitor should share with the mRNA target the same primers and most of the sequence, and the resulting PCR products should be easily discernible from one another and quantified (5,11). Usually, a single target is quantified by competitive RTPCR, and several reactions would be needed to quantitate multiple target sequences in a given RNA sample. We present here a versatile method to synthesize competitor polyA-extended RNA sequences, which are ideal for multiplex RT-PCR in which reverse transcription may be primed with oligo dT. We show that this multiplex competitive RT-PCR approach can be used to detect mRNA species as low as 103 molecules/reaction. A rapid PCR-based method to synthesize internal standards for competitive PCR has been previously described (3). Briefly, the target sequences are amplified with two primers, one conventional sense primer (Figure 1A, primer a) to be used for the subsequent experiments and a modified antisense primer (Figure 1A, primer b1). The resulting PCR products, which by design are approximately 10% shorter than the target sequences (Figure 1A), are gel purified and reamplified with a second pair of primers containing, respectively, a HindIII site (sense primer) and oligo dT21 followed by an XbaI site (antisense primer) (Figure 1B). These two PCR amplification steps can be combined, but the required primers would be quite long and there would be a risk of poor amplification. The PCR products are then digested with the two enzymes, gel purified and directionally cloned into pGem4Z (Promega, Milan, Italy) (Figure 1C). The recombinant plasmids are linearized with XbaI, and RNA synthesis is performed in vitro using a commercially available kit (Riboprobe; Promega) with minor modification to the manufacturer’s instructions (Figure 1D). Briefly, 2 μg linearized plasmid is incubated in 40 mM Tris-HCl (pH 7.9 at 25°C), 10 mM NaCl, 6 mM MgCl2, 10 mM DTT, 2 mM spermidine, 0.05% Tween( 20, 0.5 mM each ATP, GTP, CTP and UTP and 40 U T7 RNA polymerase in a final volume of 100 μL for 2 h at 37°C. The samples are heated at 95°C for 2 min and placed on wet ice. Twenty microliters of the mixture is Benchmarks