Application Progress of Massively Parallel Sequencing Technology in STR Genetic Marker Detection.

In recent years, more and more forensic genetics laboratories have begun to apply massively parallel sequencing (MPS) technology, that is, next-generation sequencing (NGS) technology, to detect common forensic genetic markers, including short tandem repeat (STR), single nucleotide polymorphism (SNP), the control region or whole genome of mitochondrial DNA (mtDNA), as well as messenger RNA (mRNA), etc., for forensic practice, such as individual identification, kinship analysis, ancestry inference and body fluid identification. As the most widely used genetic marker in forensic genetics, STR is currently mainly detected by capillary electrophoresis (CE) platform. Compared with CE platform, MPS technology has the advantages of simultaneous detection of a large number of genetic markers, massively parallel detection of samples, the polymorphism of sequence detected by NGS makes STR have the advantages of higher resolution and system efficiency. However, MPS technology is expensive, there is no uniform standard so far, and there are problems such as how to integrate MPS-STR data with the existing CE-STR database. This review summarizes the current status of the application of MPS technology in the detection of STR genetic markers in forensic genetics, puts forward the main problems that need to be solved urgently, and prospects the application prospect of this technology in forensic genetics.

[A modified method for spermatogonial cell sorting].

OBJECTIVE: To improve the method of sorting undifferentiated and differentiated spermatogonial cells by magnetic bead sorting with specific antibodies. METHODS: Using the magnetic bead sorting technique combined with Thy1 and c-Kit specific antibodies, we sorted Thy1+ and c-Kit+ cells in the testis of 7-postnatal-day male mice as undifferentiated and differentiated spermatogonia, respectively. We determined the purities of the two types of spermatogonial cells by immunofluorescence and flow cytometry, identified them via the differential expressions of Gfrα1, Plzf, c-Kit and Sohlh2 by real-time quantitative PCR, and cultured the Thy1+ cells primarily. RESULTS: The purities of the Thy1+ and c-kit+ cells were as high as (85.65 ± 8.35)% and (89.40 ± 2.77)%, respectively (P < 0.01). The relative expressions of the Gfrα1 and Plzf genes were 9.47 ± 1.29 and 4.40 ± 0.59 times higher in the Thy1+ than in the c-Kit+ cells, and those of the kit and sohlh2 genes 7.38 ± 1.07 and 3.88 ± 0.28 times lower in the former than in the latter (P < 0.01). After primary culture, the cells were seen in a normal state, proliferating smoothly with the characteristics of the proliferation of spermatogonial stem cells. CONCLUSIONS: The magnetic bead sorting technique with Thy1 and c-Kit specific antibodies can be used to effectively identify undifferentiated and differentiated spermatogonia and culture undifferentiated Thy1+ cells in vitro.

A novel elastic liposome for skin delivery of papain and its application on hypertrophic scar.

This study aims to investigate the therapeutic effects of papain elastic liposomes (PEL) on hypertrophic scar through topical application. PEL were prepared using the reverse-phase evaporation method and optimized by response surface methodology. The transdermal absorption of optimized PEL was tested by vertical Franz diffusion cells in vitro. The effects of PEL were investigated in rabbit model of hypertrophic scar in vivo, histological analysis and scar-related proteins were detected to reveal potential scar repair mechanism. The best formulation of PEL had EE (43.8±1.4%), particle size (100.9±2.2nm), PDI (0.037±0.003), zeta potential (-26.3±1.3mV), and DI (21.9±3.1). PEL gave the cumulative amounts and steady state fluxes in the receiver solution of 381.9±32.4µg/cm2, 11.4±1.5µg/cm2/h, and showed drug deposition in skin of 19.1±3.2% after 24h. After topical application, the scar elevation index, microvascular density, and collagen fiber were significantly decreased with regular arrangement. The expressions of TGF-ß1, P-Smad-3, P-NF-κB p65, and P-IKBa in hypertrophic scar were significantly down regulated in contrast with those in model group. PEL were proven as an excellent topical preparation for hypertrophic scar treatment.

[Correlation between five RNA markers of rat's skin and PMI at different temperatures].

OBJECTIVE: To explore the correlation between postmortem interval (PMI) and five RNA markers of rat's skin--ß-actin, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), 18S ribosomal RNA(18S rRNA), 5S ribosomal RNA (5S rRNA), and microRNA-203 (miR-203), at different temperatures. METHODS: Eighteen SD rats were randomly divided into three environmental temperature groups: 4 °C, 15 °C and 35 °C, respectively. Skin samples were taken at 11 time points from 0 h to 120 h post-mortem. The total RNA was extracted from the skin samples and the five RNA levels were detected by real-time fluorescent quantitative PCR. Proper internal reference was selected by geNorm software. Regression analysis of the RNA markers was conducted by GraphPad software. RESULTS: 5S rRNA and miR-203 were most suitable internal references. A good linear relationship between PMI and RNA levels (ß-actin and GAPDH) was observed in two groups (4 °C and 15 °C), whereas the S type curve relationship between the expression levels of the two markers (ß-actin and GAPDH) and PMI was observed in the 35 °C group. The partial linear relationship between 18S rRNA and PMI was observed in the groups (15 °C and 35 °C). CONCLUSION: Skin could be a suitable material for extracting RNA. The RNA expression levels of ß-actin and GAPDH correlate well with PMI, and these RNA markers of skin tissue could be additional indice for the estimation of PMI.

[Relationship between PMI and relative expression of myocardial various RNAs in rats died of different causes].

OBJECTIVE: To observe the changes of relative expression of myocardial various RNAs in rats died of different causes and their relationship with PMI. METHODS: The rat models were established in which the rats were sacrificed by broken neck, asphyxia, and hemorrhagic shock. Total RNAs were extracted from myocardium. The quantitative real time PCR was used to calculate threshold cycle values of RNAs including glyceraldehyde-3-phosphate dehydrogenase (GAPDH), beta-actin, inducible nitric oxide synthase (iNOS), hypoxia-inducible factor-1 (HIF-1), tumor necrosis factor-alpha (TNF-alpha), interleukin-6 (IL-6), and U6 small nuclear RNA (U6 snRNA) and to study the changes of the relative expressions of various indexes with PMI. RESULTS: U6 snRNA with stable expression level could be used as appropriate internal control. In the early PMI, the relative expression of GAPDH, HIF-1, iNOS, TNF-alpha, and IL-6 more characteristically increased in groups of asphyxia and hemorrhagic shock than in group of broken neck, but the quantity of beta-actin decreased in all groups. In the late PMI, all the relative expressions significantly declined in correlation with the degradation of RNA. CONCLUSION: The characteristic changes of each RNA expression can be used as references to estimate PMI in deaths by different causes.

[Application of molecular autopsy in sudden death caused by inherited arrhythmia].

Sudden cardiac death (SCD) refers to sudden stop of breath and heartbeat and death within one hour caused by underlying cardiac diseases. Clinical manifestation of inherited arrhythmia is lethal arrhythmia without gross cardiac lesions, which can lead to SCD. The autopsy and pathological examination are difficult to identify the cause of death. Fatal mechanism of inherited arrhythmia is the change in the genes encoding for cardiac ion channel protein, which causes the dysfunctions of cardiac electrical activity. It is very important to detect genetic mutation by the technique of molecular biology in negative autopsy. This review presents the latest research on the relation between SCD and inherited arrhythmia, and the application of molecular autopsy used in identifying SCD due to inherited arrhythmia and its candidate gene.