Comparing semi-automated microscopic sperm counting to DNA quantitation using qPCR to assess DNA quantities for forensic analysis
OA Version
Citation
Abstract
There are many options when it comes to performing DNA quantitation. One of the most common is performing a quantitative polymerase chain reaction (qPCR), though cell enumeration may also provide an alternate pathway. In this study, a traditional qPCR approach was compared to a semi-automated cell enumeration method using the MetaSystems Metafer software to assess if there were differences in the DNA estimations between the two. A 1:100 and 1:500 dilution were prepared from four different aliquots of the same semen sample. Twelve samples of each were quantitated using cell enumeration and twelve using qPCR. Two procedure reviews were also performed to assess reproducibility between Metafer scans and the validity of the dilution and extraction procedure. The results revealed both inter- and intra-method variation which may have arisen for a variety of reasons, including lack of reproducibility in semen pipetting, method-specific considerations, and human error. The differences in DNA quantities between the two methods were found to be statistically significant (p < 0.0001 for all except the 1:100 dilution from aliquot C where p < 0.01 and the 1:100 dilution from aliquot D where p < 0.0005). The average ratio of qPCR quantities to cell enumeration quantities was 3.69 for the 1:100 dilution and 2.15 for the 1:500 dilution.
Description
License
Attribution 4.0 International