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Reverse transcription-PCR (RT-PCR), combined with an endogenous and exogenous standard, is used extensively to quantify specific mRNAs, but few studies have evaluated the reproducibility of these assays (1)(2)(3)(4)(5)(6)(7). Increased expression of the epidermal growth factor receptor (EGFr) has been observed in several human cancers and has been suggested to predict tumor progression (8)(9)(10)(11). Quantitative analysis for the EGFr mRNA using competitive RT-PCR has been described (12)(13)(14), but the analytical imprecision has not yet been documented systematically.A primer pair was designed to flank the first intron of the human EGFr gene (15)(16). The 350-bp cDNA fragment was amplified as described below, using the following primers: primer I (5′-GAC CCT CCG GGA CGG-3′), spanning positions 191–205; and primer II (5′-GGC ATA GGA ATT TTC GTA GTA CAT AT-3′), spanning positions 515–540 and cloned into a pCRTM II vector (TA Cloning System, Invitrogen). The “EGFr” plasmid was purified using a Qiagen plasmid midi kit (Qiagen, Inc.) and cleaved by Bgl II (Pharmacia Biotech). A 69-bp deletion was obtained by PCR (see below) using the linear plasmid as template, primer III (5′-GAC CCT CCG GGA CGG CCG GGC AAG GCA CGA GTA ACA AGC TCA CGC AGT TGG 3′), and primer II (Fig. 1⇓ A). The PCR product was cloned into a pCR II vector, purified, and cleaved as above. The PCR products of the EGFr and standard were sequenced on both strands, confirming their identity and the absence of PCR errors (data not shown).To synthesize the RNA standard, 5 μg of the linear plasmid (containing the 69-bp deletion) was incubated with 100 units of SP6 RNA polymerase according to the manufacturer’s protocol (Promega Corp.). Fifty …

clinical chemistry

Quantitative Analysis of the Human Epidermal Growth Factor Receptor Messenger RNA Using Reverse Transcription-PCR: A Methodological Study of Imprecision