There is significant interest in the use of miRNA analysis for forensic body fluid identification. Demonstrated co-extraction and detection in DNA extracts could make the use of miRNAs a more streamlined molecular body fluid identification method than other RNA-based methods. We previously reported a reverse transcription-quantitative PCR (RT-qPCR) panel of eight miRNAs that classified venous and menstrual blood, feces, urine, saliva, semen, and vaginal secretions using a quadratic discriminant analysis (QDA) model with 93% accuracy in RNA extracts. Herein, miRNA expression in DNA extracts from 50 donors of each body fluid were tested using the model. Initially, a classification rate of 87% was obtained, which increased to 92% when three additional miRNAs were added. Body fluid identification was found to be reliable across population samples of mixed ages, ethnicities, and sex, with 72–98% of the unknown samples classifying correctly. The model was then tested against compromised samples and over biological cycles, where classification accuracy varied, depending on the body fluid. In conclusion, we demonstrated the ability to classify body fluids using miRNA expression from DNA extracts, eliminating the need for RNA extraction, greatly reducing evidentiary sample consumption and processing time in forensic laboratories, but acknowledge that compromised semen and saliva samples can fail to classify properly, and mixed sample classification remains untested and may have limitations.

Current processing techniques for harvesting DNA from osseous elements are destructive, and success rates vary widely. When skeletal elements are homogenized into a fine powder, endogenous DNA may be subjected to fragmentation, and the likelihood of introducing co‐purified inhibitory substances to the sample increases. While a limited number of articles in the relevant literature have challenged the status quo of pulverization, powdering hard tissue samples before DNA isolation continues to be standard practice in the forensic and ancient DNA communities. In this work, we have developed and optimized an alternative front‐end processing method for demineralizing and slicing cortical bone using aged and weathered bovine skeletal samples as a model for degraded human bone. Additionally, this study evaluated the enzymatic digestion of demineralized bone slices using collagenase I as a powder‐free alternative for cell isolation. The efficacy of pulverization, demineralized slices, and collagenase‐digested demineralized slices was assessed via DNA quantitation and STR profile data. The combined treatment incorporating collagenase digestion of demineralized slices did not improve the recovery of endogenous DNA yields or STR profile development; however, profiles developed from demineralized slices retained a significantly greater percentage of expected alleles and higher peak heights than samples processed with conventional powdering and organic chemistry extraction. By eliminating pulverization of bone and employing modified silica‐based extraction chemistry, the susceptibility to inhibitory and competitive effects from native and foreign components often found in degraded skeletal remains can be minimized when utilizing total demineralization of small fragments with subsequent slicing before lysis.

Advances in DNA technology have significantly enhanced the forensic community's ability to develop genetic profiles from unidentified human skeletal remains. However, sampling requires mechanical grinding of hard tissues before DNA isolation. This processing can compromise genetic profiles, particularly in aged bones. We compared the industry‐standard pulverization method with an alternative powder‐free preparation involving prolonged demineralization and subsequent slicing of 19th‐century cortical bone. Data from DNA quantification, STR genotyping, and targeted SNP sequencing were used to evaluate powdered samples versus demineralized slices from paired human bones. Average human DNA yields for pulverized samples and demineralized slices were 0.032 ng and 0.692 ng, respectively. Demineralized slices recovered more amplifiable DNA than traditional homogenization methods ( p < 0.05). No pulverized samples produced STR profiles, whereas demineralized slices from the same bone samples yielded partial profiles. Samples underwent DNA repair, library preparation, and hybridization capture using the FORensic Capture Enrichment (FORCE) panel. Applying low‐coverage (1X) analysis of high‐throughput sequencing (HTS) data, demineralized slices outperformed those prepared by traditional pulverization methods ( p < 0.05) and substantially increased the information recovered compared with conventional STR analysis methods. Based on HTS data from pulverized samples, DNA fragment length ranged from 27 to 95 bp, and FORCE SNP recovery was 33.23%. In contrast, for demineralized slices, DNA fragment length ranged from 85 to 114 bp, and FORCE SNP recovery was 83.24%. The required reagents and equipment are typically available in forensic labs, and the workflow outlined herein significantly increases the success of DNA recovery from challenging skeletal samples.

Background: Cervical cancer remains a major global health concern, with disparities in screening access and outcomes, particularly among younger women in the United States. Although high-risk human papillomavirus infection is the primary etiologic factor, nongenetic and environmental contributors remain underexplored. We established the Sacred Womb Cervical Cytology Biorepository to support research on cervical cancer risk, molecular biomarkers, and gynecologic health disparities. Methods: We implemented standardized protocols for the recovery, processing, and long-term storage of residual ThinPrep Papanicolaou (Pap) specimens from women aged 21 to 65 receiving routine gynecologic care across VCU Health outpatient clinics. Core elements included an opt-out consent model, an Institutional Review Board–approved waiver for discarded cytology material, electronic health record linkage for longitudinal clinical data, and workflows for specimen recovery, aliquoting, and processing of pellet and supernatant fractions. Specimens are cryostored at −80°C with quality control checks and secure data governance. Results: The biorepository supports standardized recovery and processing of residual Pap specimens, yielding sufficient postdiagnostic volume and enabling high-quality genomic DNA extraction and metabolomic profiling. Pilot genomic and metabolomic data demonstrate that residual cervical cytology material provides adequate input for multiomic analyses, confirming feasibility and scalability. Conclusions: This protocol outlines a framework for establishing a cervical cytology biobank using residual clinical specimens. The integration of biospecimens with linked clinical and demographic data enables multidimensional research into cervical cancer etiologies and other gynecologic conditions. Impact: This resource supports molecular epidemiology studies aimed at identifying biomarkers, understanding cervical cancer progression, and addressing reproductive health disparities in diverse populations.