Before proceeding with the analysis of the samples, all the working surfaces were treated with disinfectant and antifungal solutions, left to act for about 30 minutes before cleaning. Furthermore, to limit contamination as much as possible, an appropriate support (MicroAmp® Splash Free 96-Well Base, Applied Biosystems) was used to prevent contact between the plate containing the samples and the working surfaces.
The “Quantifiler Duo DNA Quantification Kit” used for the amplications of the previously extracted DNA samples includes the reagents necessary for amplification, identification, and quantification of one specimen of human DNA [uno specifico DNA umano] and one specimen of human male DNA, as previously reported. All the reagents present in the kit are optimized for use with the Applied Biosystems 7500 Real-Time PCT System apparatus equipped with SDS software.
For each DNA sample and for each point of the standard curve, a reaction mixture was prepared according to the following scheme:
The necessary amounts of the components were prepared in appropriate sterile polypropylene tubes, and 23 μL of reaction were dispensed in each well [pozzetto] of a 96-well plate for Real-Time (MicroAmp Optical 96-well reaction plate, Part number N801-0560, Applied Biosystems). Subsequently, 2 μL of standard DNA, 2 μL of DNA to be analyzed, and 2 μL of control (NTC) were added to each well to obtain the final reaction volume of 25 μL/sample.
Before being processed, the plate was centrifugated at 300 rpm for about 30 seconds in order to remove any air bubbles in the wells.
The samples, loaded onto the 96-well plate, were analyzed according to the following scheme:
where: S stands for Standard and U for Unknown. All the DNA samples were analyzed in triplicate using 2 μL of volume for each reaction (total 25 μL) and are indicated with the letters A, B, C, D, E, F, G, H, I, L, and M. NC refers to a negative control sample while NTC identifies the Non Template Control, that is the PCR sample lacking amplifiable DNA. The standard curve was analyzed in duplicate.
The parameters relevant to the PCR are the following:
The standard curve was constructed via 8 noted concentrations of DNA (provided with the kit). The concentrations used run from 50 ng/μL (point 1) to 0.023 ng/μL (point 8). A duplicate for each standard point was used.
The analysis at the end of the run, performed via the SDS software, provided the results reported below.