Evaluation of real-time quantitative PCR as a standard cytogenetic diagnostic tool for confirmation of microarray...

To evaluate the use of real-time quantitative PCR (qPCR) as a diagnostic tool for follow up of abnormal microarray (aCGH) results.

qPCR was performed on 207 samples with known aCGH results to detect chromosomal abnormality and determine the capability of qPCR. Eighty-four samples were processed and the results compared with the original aCGH result and with one or more of the alternative follow-up methods: aCGH, fluorescence in situ hybridization (FISH), or karyotyping. A separate cohort of 107 samples was used to determine critical threshold values for qPCR. A further 16 samples were assessed in reproducibility and sensitivity studies.

All qPCR findings were consistent with the original aCGH results, and qPCR was found to be a superior follow-up method compared to FISH and karyotyping. Critical threshold values were also determined from this study.

In this study, qPCR analysis identified all copy number changes. qPCR is an accurate, rapid, reliable, and inexpensive technique for confirming copy number changes, and for determining the inheritance of such abnormalities to aid interpretation of results. We also present the critical threshold values required for qPCR as a practical tool. This technique has now been successfully implemented as part of the clinical diagnostic service within our laboratory.