Qiasymphony apparatus

This study was performed using samples collected for the REDS-III Brazil SCD cohort study (9) eligible participants were randomly selected that included institutions in four Brazilian states: São Paulo (Hospital das Clínicas), Minas Gerais (Hemominas), Rio de Janeiro (Hemorio) and Pernambuco (Hemope).
The study was approved by the local ethical review committee of participating institutions,namely, the Pro-Sangue foundation, Hemominas foundation, Hemope foundation and Hemorio blood bank. Also, the study was approved by the REDS-II collaborating centers (Blood Systems Research Institute/University of California at San Francisco, San Francisco, CA) and data-coordinating center (Westat, Inc.) in the United States.
The samples were collected in an EDTA tube, centrifuged at 3500rpm, and plasma was separated from cells. Both components were frozen and shipped to the central laboratory at the University of Sao Paulo for further testing.
DNA extraction was performed using the QIAsymphony apparatus (Qiagen, Germany) and the QIAsymphony DNA Mini Kit (Qiagen, Germany), following the manufacturer's instructions and protocol.

All serum samples tested positive with more than 1 ml available were analyzed. Storage at -80°C before PCR did not exceed 2 years. After thawing, DNA from 1 mL of serum was extracted using the Qiasymphony DSP virus/Pathogen Mini kit (Qiagen) and a Qiasymphony apparatus (Qiagen), eluted in 85 μL, and tested in duplicate using the 28S rDNA PCR assay previously reported (Challier et al., 2004 (link)). Primer and probe concentrations were set at 0.3 and 0.1 μM in the 480 probe Master (Roche), respectively, and the PCR assay was performed in a LightCycler 480 instrument (Roche).
The results were expressed in quantification cycles (Cq), with higher values indicating less targeted DNA in the sample. Positivity was defined by at least one of the two duplicates having Cq ≤ 45 cycles. The mean value of the duplicates was retained for further comparisons when both were positive and the single value when one replicate was positive alone. DNA extraction and amplification yields were assessed using the Simplexa Extraction and Amplification Control Set (Focus Diagnostics, Cypress, CA, United States) as an internal control (IC). The PCR assay was performed blind to interpretation of the GM results (true or false positives) and to the clinical classification.