Reliability of PCR-based detection of occult tumour cells: lessons from real-time RT-PCR

Abstract

For the molecular detection of rare tumour cells in clinical samples, real-time reverse transcription-polymerase chain reaction (RT-PCR) offers two important advantages over conventional RT-PCR assays: the results are quantitative and, perhaps more importantly, it facilitates exact sensitivity controls on a per sample basis as well as exact comparison of different assay protocols. We report here on quantitative results obtained with different protocols for RNA isolation and cDNA synthesis for amplification of beta2-microglobulin transcripts using the light cycler system. Furthermore, housekeeping gene-specific PCRs were compared with PCRs specific for an artificial transcript (internal standard) detected simultaneously at a level comparable to the wild-type sequence. Artificial tyrosinase transcripts derived from a vector construct stably transfected into a human lymphoma cell line were used as a model to test the usefulness of artificial internal standards as an alternative to housekeeping genes. The highest RNA yields were obtained using a combination of phenol-chloroform extraction and the High Pure RNA Isolation Kit. Analysing beta2-microglobulin transcript-specific RT-PCRs, the highest sensitivity was obtained for cDNAs generated with Omniscript reverse transcriptase and oligo-p(dT)15 primer. Regarding patient blood samples, RT-PCRs specific for beta2-microglobulin, porphobilinogen deaminase and artificial tyrosinase transcripts provided quantitative data for all, for 18 out of 21, and for 10 out of 21 samples, respectively. Quantification of beta2-microglobulin transcripts by the light cycler system defined the protocol revealing the highest cDNA quality. Comparisons of quantitative data from RT-PCRs specific for beta2-microglobulin, porphobilinogen deaminase and artificial tyrosinase transcripts enabled us to determine a close range for crossing points within which sufficient cDNA quality can be guaranteed, even for the detection of rare transcripts. PCRs specific for the artificial internal standard are ideally suited for cDNA quality assessment on a per sample basis.

Quantitative Detection of Circulating Tumor Cells in Cutaneous and Ocular Melanoma and Quality Assessment by Real-Time Reverse Transcriptase-Polymerase Chain Reaction

“…The quantification of serial plasmid dilutions yielded linear crossing point increases over a range of 7 logs (data not shown; Ref. 40).…”

“…The crossing point values of this method are linear over 7 logs (40). The limit of detection was tested for tyrosinase expression in five experiments and 10 melanoma cells of the cell line SK-MEL 24 in 10 ml of blood (data not shown).…”

“…With conventional or semiquantitative RT-PCR, however, the determination of cDNA quality by means of detection of housekeeping genes is by far not optimal. Quality control by using detection of small amounts of plasmid or foreign cellular standards (40) admixed with the sample is also not an optimal control, because potential damage before admixing the sample with this type of standard cannot be controlled.…”

Real-time reverse transcription PCR (qRT-PCR) and its potential use in clinical diagnosis

“…The problem with this approach is that the highest dilutions provide information about the variability of the assay at those very low target copy numbers. qRT-PCR facilitates the inclusion of exact sensitivity controls on a per sample basis [53] and, clearly, PCR findings, positive or negative, are questionable if they are not supported by the associated data demonstrating the overall sensitivity of the assay applied [44]. Therefore it is acceptable to omit the highest dilutions if the target copy numbers are well above that threshold number.…”

PCR-based Calibration Curves for Studies of Quantitative Gene Expression in Human Monocytes: Development and Evaluation

“…Measurements of specific mRNAs through quantitative RT-PCR poses numerous problems; in fact, it requires the isolation of intact mRNA species, quantitative reverse transcription, and quantitative PCR amplification (4,32,33 ). In the present study, the first step focused on the development and evaluation of PCR amplicon-based calibrators to generate calibration curves that could be used to quantify specific target mRNAs.…”