Evaluation of reverse transcription yield of RNA standards and forensic samples based on droplet digital PCR.

RNA analysis has shown great value in forensic science, such as body fluids and tissue identification, postmortem interval estimation, biological age prediction, etc. Currently, most RNA follow-up experiments involve reverse transcription (RT) procedures. It has been shown that the RT step is variable and has a greater impact on subsequent data analysis, especially for forensic trace samples. However, the pattern of variation between different RNA template inputs and complementary DNA (cDNA) yield is unclear. In this study, a series of 2-fold gradient dilutions of RNA standards (1 µg/µL - 0.24 ng/µL) and forensic samples (including blood samples, saliva samples, bloodstains, and saliva stains) were reverse-transcribed using EasyQuick RT MasterMix. The obtained cDNA was quantified by droplet digital PCR (ddPCR) to assess the RT yield of the ACTB gene. The results showed that the 125 ng RNA template had the highest RT yield in a 10 µL RT reaction system with the selected kit. For all stain samples, the RT yield improved as the amount of RNA template input increased since RNA quantities were below 125 ng. As many commercialized reverse transcription kits using different kinds of enzymes are available for forensic RNA research, we recommend that systematic experiments should be performed in advance to determine the amount of RNA input at the optimum RT yield when using any kit for reverse transcription experiments.

Visual identification for species and sex derived from bloodstain based on phosphate-mediated isothermal amplification colorimetric system.

Species and sex confirmation of the biological specimen play a crucial role in crime investigation. However, the specimen found in the scene is always trace quantity, which is hard to be analyzed by current methods. Moreover, the time-consuming DNA extraction, sophisticated apparatus, and complex data processing make it difficult to satisfy the demand of speediness and convenience for point-of-care tests. In this study, we first exhibit a phosphate-based visual system for field-based species and sex identification derived from trace bloodstain. By introducing phosphate ion-based colorimetry into loop-mediated isothermal amplification (LAMP) for result interpretation, not only the bloodstain can be directly submitted to mitochondrial variant amplification owing to the enhanced amplification efficiency by pyrophosphate ion hydrolyzation, but also the colorimetric signal can be recognized by the naked eye for result output within 30 min through molybdophosphate generation. Aerosol contamination, the major conflict of LAMP, has been solved once and for all by integrating uracil-DNA glycosylase into this system that still holds on a constant temperature. As a demonstration, cytochrome b and Y-chromosomal amelogenin are employed to identify species and sex respectively, which has achieved a highly sensitive and specific distinguishability under a strong interferential background. Accurate results can be obtained from both the simulative degraded and dated specimen, which indicates that this novel system may serve as a promising tool in forensic practice.

Circadian metabolites for evaluating the timing of bloodstain deposition: A preliminary study.

Metabolites, as products of cellular metabolism, can provide a wealth of biological information and are less susceptible to degradation than other biomarkers due to their low molecular weight. Due to these properties, metabolites can be used as valuable biomarkers for forensic investigations. Knowing the timing of deposition of bloodstain could help to reconstruct crime scenes, draw conclusions about the time of the crime, and narrow down the circle of possible suspects. Previous studies have indicated that the concentration of some metabolites in blood is subject to circadian changes. However, the circadian metabolites of bloodstains have been still unclear. A total of sixty-four bloodstain samples were prepared under real conditions in three time categories (morning/noon (09:00 h ∼ 17:00 h), afternoon/evening (18:00 h ∼ 23:00 h) and night/early morning (24:00 h ∼ 08:00 h)). Fifty metabolites of bloodstains with significant differences were identified in the three time categories. Twenty-eight of these metabolites exhibited significant circadian changes. Finally, three independently contributing circadian metabolites were selected to build the logistic regression model, with an area under the curve of 0.91, 0.84 and 0.87 for the prediction of bloodstain deposition time in the morning/noon, afternoon/evening and night/early morning, respectively. The study indicated that circadian metabolites can be used for evaluating the timing of bloodstain deposition. This would provide a valuable perspective for analyzing the deposition time of biological traces in forensic investigations.

Downregulation of the expression of galanin impairs erectile function in hypoandrogenic rats.

Background: The relationship between galanin and erectile function under low androgen levels is still unclear. Aim: To explore whether a low testosterone level damages the erection of a rat by regulating the expression of galanin and GalR in penile cavernous tissue. Methods: Thirty-six male Sprague-Dawley rats, 8 weeks of age, were randomly grouped as follows (n = 6): control, castration, castration + testosterone replacement, control + transfection, castration + transfection, and castration + empty transfection. At 4 weeks after castration, rats in the transfection group were injected with lentivirus carrying the targeting galanin gene (2 × 108 TU/mL, 10 µL) in the corpus cavernosum. After 1 week of injection, the intracavernosal pressure (ICP), mean arterial blood pressure (MAP), nitric oxide (NO), serum testosterone concentration, galanin, GalR1-3, ROCK1, ROCK2, and p-eNOS/eNOS in the rat penile tissues were evaluated. Outcomes: ICPmax/MAP and the expression of galanin in the corpus cavernosum in castrated rats were obviously decreased as compared with those in the control rats. Results: The castrated rats showed remarkably lower ICPmax/MAP, galanin, GalR1-3, p-eNOS/eNOS, and NO content and markedly higher ROCK1 and ROCK2 in penile tissues than the control group (P < .05). The transfected rats administrated with LV Gal had obviously higher ICPmax/MAP, p-eNOS/eNOS, and NO content and less ROCK1 and ROCK2 protein expression in the corpus cavernosum when compared with the castration group (P < .05). Clinical Translation: Upregulating the expression of galanin in the penile corpus cavernosum might be a novel method of treating erectile dysfunction caused by a low androgen level. Strengths and Limitations: The conclusions obtained in the animal experiments need to be confirmed in human data. Conclusion: The erectile function of hypoandrogen rats might be inhibited by downregulating the level of galanin and GalR1-3, upregulating ROCK1 and ROCK2 levels, and inhibiting the eNOS/NO signaling pathway in penile corpus cavernosum.