Reliability of PCR-based detection of occult tumour cells: lessons from real-time RT-PCR.
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.
Max
N
N
Department of Medicine III, University Hospital Benjamin Franklin, Free University Berlin, Hindenburgdamm 30, 12200 Berlin, Germany. max@ukbf.fu-berlin.de
Willhauck
M
M
Wolf
K
K
Thilo
F
F
Reinhold
U
U
Pawlita
M
M
Thiel
E
E
Keilholz
U
U
D018076:DNA, Complementary / Q000096:biosynthesis / Q000235:genetics; D006801:Humans; D011163:Hydroxymethylbilane Synthase / Q000097:blood / Q000235:genetics; D008545:Melanoma / Q000097:blood / Q000175:diagnosis* / Q000201:enzymology / Q000235:genetics*; D014442:Monophenol Monooxygenase / Q000097:blood / Q000235:genetics; D018365:Neoplasm, Residual / Q000097:blood / Q000175:diagnosis / Q000235:genetics; D009360:Neoplastic Cells, Circulating; D016133:Polymerase Chain Reaction / Q000379:methods*; D011786:Quality Control; D012334:RNA, Neoplasm / Q000097:blood* / Q000235:genetics / Q000302:isolation & purification; D012015:Reference Standards; D015203:Reproducibility of Results; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013997:Time Factors; D001613:beta 2-Microglobulin / Q000097:blood / Q000235:genetics
D018076:DNA, Complementary; D012334:RNA, Neoplasm; D001613:beta 2-Microglobulin; D014442:Monophenol Monooxygenase; D011163:Hydroxymethylbilane Synthase