Optimization of PCR amplification for B-and T-cell clonality analysis on formalin-fixed and paraffin-embedded samples |
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Authors: | Laszlo Bereczki Gyongyi Kis Eniko Bagdi Laszlo Krenacs |
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Institution: | (1) Laboratory of Tumor Pathology and Molecular Diagnostics, Institute for Biotechnology, Bay Zoltan Foundation for Applied Research, Derkovits fasor 2., H-6726 Szeged, Hungary |
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Abstract: | In many cases, particularly in retrospective studies, only formalin-fixed and paraffin-embedded (FFPE) tissue samples are
available for molecular studies. DNA recovered from FFPE tissues generally consists of fragmented small target sequences with
chemical alterations. Clonality analysis is not easy on FFPE samples, in fact, it requires even more experience than that
of performed on fresh samples or is more complicated than most genomic PCR amplifications for somatic genes. In our study,
we have performed a multi-parameter PCR evaluation investigating immunoglobulin heavy chain gene (IgH) and T-cell receptor
gamma gene (TCRy) rearrangements on non-purified crude lysates of FFPE samples, in order to establish the significance of
different variables on performance of PCR amplification. The results showed that a slight decrease in the concentration of
primers in combination with a slight increase in MgCl2 andTaq polymerase concentrations, as well as the use diluted crude template and a standard amount of dNTPs can be the modifications
of choice while adjusting IgH and TCRy clonality tests on poor quality DNA FFPE samples. Using our improved protocol, 74%
(17/23) of the tested B-cell lymphomas and 68% (31/46) of the tested T-cell lymphomas demonstrated monoclonal PCR product,
proving the applicability of our optimized method. Our experience may be of help during the optimization process in technically
difficult cases as well as to determine which parameters and how should be changed to minimize false-negative and false-positive
results.
Supported in part by the OTKA Research Fund (T046663 KON) and the Hungarian Academy of Sciences, Bolyai Janos Research Fellowship
(E.B.). |
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Keywords: | PCR optimization B- and T-cell clonality archived tissues |
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