Quantifiler trio dna quantification kit

DNA extraction and STR analyses were by the Denver Police Department Crime Laboratory DNA Unit using validated forensic standard operating procedures [16 (link),17 (link)]. Samples were collected into GeneAmp thin-walled reaction tubes (0.5 ml; Applied Biosystems, Carlsbad, CA, USA). Lymphocyte DNA was extracted manually using the FFPE Tissue kit (Qiagen in USA). Total human and male DNA were assessed with the Quantifiler Trio DNA Quantification Kit (Applied Biosystems, Carlsbad, CA, USA). DNA was extracted using the RecoverAll Total Nucleic Acid Isolation Kit for FFPE Tissues (Applied Biosystems, Carlsbad, CA, USA). The number of tumor cells microdissected per sample was automatically recorded by the Arcturus XT system.

After the extraction, all samples were quantified with the Quantifiler® Trio DNA Quantification Kit (Applied Biosystems) following the manufacturer’s instructions (Vernarecci et al., 2015 (link)). Quantitative PCR (qPCR) was performed in double using QuantStudio 5 Real-Time PCR System (Applied Biosystems) and data were analyzed using the HID Real-Time PCR Analysis Software v1.3 using the default settings. The quantification data obtained from the small autosomal probe of the Quantifiler Trio Kit was used for determining the concentration of aDNA.
Y-chromosome short tandem repeats amplification was performed with the Yfiler® Plus PCR Amplification Kit (Applied Biosystems), including the 27 STRs loci DYS19, DYS385a/b, DYS389I/II, DYS390, DYS391, DYS392, DYS393, DYS437, DYS438, DYS439, DYS448, DYS456, DYS458, DYS635, YGATAH4, DYS460, DYS481, DYS533, DYF387S1a/b, DYS449, DYS518, DYS570, DYS576, and DYS627, on a Veriti® 96 Well Thermal Cycler System (Applied Biosystems) and following the manufacturer’s instructions. PCR products were electrophoresed on a SeqStudio Genetic Analyzer (Applied Biosystems) and the fragment analysis was performed by GeneMapper® ID-X Software v1.6 (Applied Biosystems). Only peaks above 50 RFU were considered and replicates were performed for all ancient samples.

The quantity of DNA extracted from both samples (pre- and post-vasectomy stains) and blood was determined by real-time PCR using the Quantifiler Trio DNA Quantification Kit (Applied Biosystems, USA) according to the manufacturer's instructions²⁸ . The reactions were run on an ABI PRISM 7500 Real-Time PCR system (Applied Biosystems, USA). The data were analyzed using the HID Real-Time PCR Analysis Software v 1.2 (Applied Biosystems, USA).

DNA was obtained from peripheral blood using the Prep-Filer Express BTA™ Forensic DNA extraction kit. For this purpose, the AutoMate Express DNA extraction system was used according to the supplier’s instructions (Applied Biosystems, Foster City, CA, USA). Next, DNA was quantified with the Quantifiler^® Trio DNA quantification kit in a 7500 Applied Biosystems Real-Time PCR system (Applied Biosystems, Foster City, CA, USA).

DNA extraction was conducted on CVL pellets using the MagNAPure LC DNA Isolation Kit I (Roche Applied Science, Indianapolis, IN, USA), as instructed by the manufacturer. The Human Y‐chromosome Quantification Kit (PrimerDesign Ltd, Chandler's Ford, UK) was used to detect a TSPY1 gene region present on the Y‐chromosome within the extracted DNA. The Y‐chromosome primer/probe mix and the PrecisionFAST™ Mastermix were used according to the manufacturer's instructions (PrimerDesign Ltd). Amplification was conducted on the Applied Biosystems^® QuantStudio™ 5 real‐time (RT)‐PCR System (Thermo Fisher Scientific, Waltham, MA, USA). Detection of YcDNA within vaginal specimens indicated semen exposure within 15 days before genital sampling [15, 16]. The Quantifiler™ Trio DNA Quantification Kit from Applied Biosystems™ (Thermo Fisher Scientific) was used to quantify YcDNA concentrations in YcDNA+ CVL specimens as outlined in the manufacturer’s protocol. The assay simultaneously quantified the total amount of amplifiable human DNA and human male DNA in 10 µL of the sample.

Anonymous DNA extracts from nine presumably degraded tissue samples were used. The extraction method was the BioRobot EZ1 (Qiagen) using the EZ1 DNA Tissue Kit (Qiagen) according to manufacturers recommendations. The extracts had been stored for about two years in a freezer prior to quantification and analysis. The Quantifiler®; Trio DNA Quantification Kit (Applied Biosystems), with an 80 bp small autosomal target and 214 bp large autosomal target, and PowerQuant^TM System (Promega), with an 84 bp small autosomal target and 294 bp large autosomal target, were used for quantification. Both kits confirmed that the tissue samples were degraded to different degrees (degradation index in Table 2). The PowerPlex®; ESX 17 Fast System (Promega) was used for STR amplification.

DNA quantification was performed using the Quantifiler™ Trio DNA Quantification Kit on the QuantStudio 5 Real-Time PCR System for Human Identification (Applied Biosystems), following the manufacturer’s protocol. DNA samples with quantification values equal to or greater than 0.015 ng/μl in T0 and T1 or in T0 and T2 were chosen to be amplified by multiplex PCR. This choice was made in order to be able to compare the profiles obtained before and after the use of the alcohol-based hand sanitizer.
Seventy-six out of 120 samples were amplified using the GlobalFiler® PCR Amplification Kit (Thermo Fisher Scientific) in accordance with the manufacturer’s recommendations with the standard 29 cycles on the Veriti™ 96-Well Thermal Cycler (Applied Biosystems) instrument. Amplified products were separated and detected on the SeqStudio™ Genetic Analyzer (Applied Biosystems). Data collection and fragments analysis was conducted using GeneMapper® ID-X v 1.6 (Thermo Fisher Scientific) with an analytical threshold set to 100 relative fluorescence units (RFU).