Internal Control for Quality Assurance of Diagnostic RT-PCR

Reverse transcription polymerase chain reaction (RT-PCR) is gaining major importance in the field of medical diagnostics. For example, RT-PCR assays are being tested to become standard methodology for detection of occult tumor cells in peripheral blood of cancer patients. However, there is heterogeneity in the frequency of tumor cell detection, e.g., in melanoma patients as reported in the literature (1,6,9). One of the main reasons for these discrepancies is differences in methodology, and a major problem is the limited availability of controls to assess the assay’s reliability. Controls for RT efficacy and RNA quality are usually accomplished indirectly by amplification of ubiquitously expressed housekeeping genes such as glyceraldehyde-3-phosphate dehydrogenase (GAPDH), β-actin and β2-microglobulin. However, the results should be interpreted with great caution for several reasons. First, the abundance of PCR targets for housekeeping genes can lead to satisfying PCR results for housekeeping genes in samples not suitable for reliable detection of tumor targets with low mRNA expression levels. Second, the increasing number of GAPDH and β-actin retropseudogenes (7,8), which can be amplified even if mRNAspecific primers were designed, can lead to an overestimation of the RT efficacy. Third, because the expression of housekeeping genes is regulated (3,4), the level of expression may be influenced by many factors. All of these reasons limit the usefulness of housekeeping gene PCR as a reliable control of RNA quality and RT efficacy, whereas application of an internal control would provide a tool for reliable evaluation of the entire RT-PCR process on a per sample basis. We developed a model of a simple and universally applicable internal control for the RT-PCR assay system for detection of tumor cells in peripheral blood of cancer patients, in which a small defined number of control cells is added to the blood samples as soon as the sample reaches the laboratory. Because the control cells are carried throughout the whole assay procedure, they are a potent indicator of the efficacy of the RT-PCR assay system. We have evaluated the feasibility of this internal control for an RT-PCR assay to detect melanoma cells using the tissuespecific tyrosinase gene (tyr) as target gene. The Jurkat T-cell line was used as a control cell, and the unique Jurkat Tcell receptor β-variable gene (TCRBV) was used as a control gene to monitor sample processing efficacy. We added 103 Jurkat cells to blood samples from melanoma patients and to a dilution series of SK-Mel 28 cells (101–104 were added to 10 mL of healthy donor blood) before processing. Processing involved RNA extraction performed with a modified guanidinium isothiocyanate (GITC) phenol–chloroform method, and the RT performed with random hexamers and avian myeloblastosis virus (AMV) reverse transcriptase (Promega, Madison, WI, USA) as described elsewhere in detail (11). PCR was carried out as coamplification with two primer sets. One published primer pair (HTYR3 and HTYR4) specific for the tyr cDNA (10) and a second primer pair specific for the unique Jurkat TCRBV cDNA were used. The Jurkat’s specific 5′ primer (5′-TGACAGAGATGGGACAAGAAG-3′) was devised from the TCRBV region, whereas the specific 3′ primer (5′-TAACCTGGTCCCCGAACCG-3′) and the hybridization oligonucleotide probe (5′-TAGCCGAACAGGTCGAGAA-3′) were designed according to the T cell’s unique complementary determining region (CDR) 3. Two microliters of first-strand cDNA/RNA heteroduplex were diluted in 50 μL containing a final concentration of 50 pmol of each primer, 1.5 mM MgCl2, 0.2 mM dNTP, 1× PCR buffer (50 mM KCl, 10 mM Tris-HCl, pH 9.0, 0.1% Triton X-100) and 2 U Taq DNA Polymerase (Promega). The samples were overlaid with 70 μL mineral oil, and 45 cycles of amplification were carried out on a thermal cycler (Perkin-Elmer, Norwalk, CT, USA) with a hot start. Each cycle consisted of a 1min denaturation step at 94°C, a 1-min annealing step at 55°C and a 1-min extension step at 72°C. The PCR fragments were 207 and 307 bp for the tyr and TCRBV genes, respectively. Eight microliters of PCR product of the tyr and TCRBV co-amplification were separated in a 2% agarose gel (SeaKem LE agarose; FMC BioProducts, Rockland, ME, USA) and blotted onto a pos-