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Posted by macmanes on 28 Sep 2012 at 22:08 GMT

DNA Extraction

How can you be sure that there was no contamination from either persons who performed autopsy, or wet lab procedures. Were these males? How did you rule out or control for contamination? Was there a negative control?

No competing interests declared.

RE: contamination

wchan replied to macmanes on 09 Oct 2012 at 17:41 GMT

We monitored for contamination during qPCR setup by running multiple no template controls (i.e. reaction wells that contained reaction mix, primers and probes but no DNA added). Per plate, two wells were run in which reaction mix and beta-globin primers and probes were added to detect general DNA contamination. Another two wells were run in which reaction mix and DYS14 primers and probes were added to detect contamination by male DNA. Pre and post PCR tasks were conducted with separate pipettes and in separate areas. All post PCR products were in sealed plates that were discarded. To reduce the possibility of including a spurious result we also took a very conservative approach to data analysis, because harvesting autopsy specimens was conducted elsewhere and a male conducted some of the work, albeit gowned, masked and gloved, with sterilized instruments and in a sterile hood. Therefore stringent requirements were included for any result to be considered positive in the data analysis. Statistical tests were also carefully selected and used to study correlations. Please see the methods section of our manuscript for complete details. We also sought to emphasize that other studies will be necessary to determine the true prevalence of microchimerism originating from prior pregnancies for a number of reasons (see discussion).

No competing interests declared.