Yak genome database: a multi-omics analysis platform.

BACKGROUND: The yak (Bos grunniens) is a large ruminant species that lives in high-altitude regions and exhibits excellent adaptation to the plateau environments. To further understand the genetic characteristics and adaptive mechanisms of yak, we have developed a multi-omics database of yak including genome, transcriptome, proteome, and DNA methylation data. DESCRIPTION: The Yak Genome Database ( http://yakgenomics.com/ ) integrates the research results of genome, transcriptome, proteome, and DNA methylation, and provides an integrated platform for researchers to share and exchange omics data. The database contains 26,518 genes, 62 transcriptomes, 144,309 proteome spectra, and 22,478 methylation sites of yak. The genome module provides access to yak genome sequences, gene annotations and variant information. The transcriptome module offers transcriptome data from various tissues of yak and cattle strains at different developmental stages. The proteome module presents protein profiles from diverse yak organs. Additionally, the DNA methylation module shows the DNA methylation information at each base of the whole genome. Functions of data downloading and browsing, functional gene exploration, and experimental practice were available for the database. CONCLUSION: This comprehensive database provides a valuable resource for further investigations on development, molecular mechanisms underlying high-altitude adaptation, and molecular breeding of yak.

Analytical and clinical performance evaluation of a new NT-proBNP assay.

BACKGROUND: The study evaluated the performance of the Mindray N-terminal pro-B-type natriuretic peptide (NT-proBNP) in a healthy population in China, focusing on creating a reference range for future clinical applications adjusted according to different demographics. METHODS: The study measured NT-proBNP in 2277 healthy individuals. We analyzed age and sex-stratified data, performed precision, accuracy, linearitcvy, and detection limit studies, and evaluated method comparison and consistency between Roche and Mindray assays on 724 serum samples. We used Excel 2010, Medcalc, and GraphPad Prism 9. RESULTS: In males, the 97.5th centile NT-proBNP concentration at age < 45, 45 to 54, 55 to 64, 65 to 74 and ≧ 75 were 89.4 ng/L, 126 ng/L, 206 ng/L, 386 ng/L and 522 ng/L, respectively. In females, the concentration of NT-proBNP at the same age was 132 ng/L, 229 ng/L, 262 ng/L, 297 ng/L and 807 ng/L, respectively. The repeatability precision coefficient of variation (CV%) for NT-proBNP was between 0.86 and 1.65 in analytical performance. In contrast, the reproducibility precision (CV%) for NT-proBNP was between 1.52 and 3.22, respectively. The study found a bias of accuracy of 3.73% in low-value samples (concentration: 148.69) and 7.31% in high-value samples (concentration: 1939.08). The sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) of 125 ng/L were 96.6%, 92.3%, 84.2%, and 98.5%, respectively. In contrast, those of 300 ng/L were 94.0%, 98.2%, 95.7% and 97.5%, respectively. CONCLUSIONS: The Mindray NT-proBNP assay showed increased levels in both males and females with age, with higher levels in women. It performs well and aligns with manufacturer specifications. We recommend adjusting cutoff values based on demographic factors.

Single-cell transcriptome analysis and in vitro differentiation of testicular cells reveal novel insights into male sterility of the interspecific hybrid cattle-yak.

BACKGROUND: Interspecific hybridization plays vital roles in enriching animal diversity, while male hybrid sterility (MHS) of the offspring commonly suffered from spermatogenic arrest constitutes the postzygotic reproductive isolation. Cattle-yak, the hybrid offspring of cattle (Bos taurus) and yak (Bos grunniens) can serve as an ideal MHS animal model. Although meiotic arrest was found to contribute to MHS of cattle-yak, yet the cellular characteristics and developmental potentials of male germline cell in pubertal cattle-yak remain to be systematically investigated. RESULTS: Single-cell RNA-seq analysis of germline and niche cell types in pubertal testis of cattle-yak and yak indicated that dynamic gene expression of developmental germ cells was terminated at late primary spermatocyte (meiotic arrest) and abnormal components of niche cell in pubertal cattle-yak. Further in vitro proliferation and differentially expressed gene (DEG) analysis of specific type of cells revealed that undifferentiated spermatogonia of cattle-yak exhibited defects in viability and proliferation/differentiation potentials. CONCLUSION: Comparative scRNA-seq and in vitro proliferation analysis of testicular cells indicated that not only meiotic arrest contributed to MHS of cattle-yak. Spermatogenic arrest of cattle-yak may originate from the differentiation stage of undifferentiated spermatogonia and niche cells of cattle-yak may provide an adverse microenvironment for spermatogenesis.

Genome-wide comparison of DNA methylation patterns between yak and three cattle strains and their potential association with mRNA transcription.

Yak has evolved specific adaptative mechanisms to high-altitude environment. Up to date, only a few studies reported the DNA methylation in yak. In the present study, genome-wide DNA methylome and transcriptome profiles in lung, mammary, and biceps brachii muscle tissues were compared between yak and three cattle breeds (Tibetan cattle, Sanjiang cattle, and Holstein cattle). The association between differentially expressed genes (DEGs) and differentially methylated regions (DMRs) was analyzed, and the biological functions of DEGs potentially driven by DMRs were explored by KEGG enrichment analysis. Finally, we found that yak-specific DMRs-driven DEGs were mainly involved in neuromodulation, respiration, lung development, blood pressure regulation, cardiovascular protection, energy metabolism, DNA repair, and immune functions. The higher levels of the key genes associated with these functions were observed in yak than in cattle, suggesting that DNA methylation might regulate these genes. Overall, the present study contributes basic data at the DNA methylation level to further understand the physiological metabolism in yak.

Dynamic alterations in yak (Bos grunniens) rumen microbiome in response to seasonal variations in diet.

Rumen microorganisms play important roles in the healthy growth of yaks. This study investigated changes in yak rumen microbiome during natural grazing at the warm seasons and supplementary feeding at cold seasons. High-throughput sequencing of 16S rRNA and metagenome analysis were conducted to investigate the structures and functions of yak rumen microbial communities. The results indicated that Bacteroidetes and Firmicutes were the most abundant phyla. In addition, Bacteroidetes might play a more important role than Firmicutes during the supplementary feeding stage (spring and winter), but less during natural grazing stage (summer and autumn). KEGG analysis showed that the amino sugar and nucleotide sugar metabolism, glycolysis/gluconeogenesis, pyruvate metabolism, starch and sucrose metabolism, and fructose and mannose metabolism were the main pathways in the microbial community, which were significantly different between seasons. The carbohydrate-active enzymes (CAZyme) annotation revealed that cellulose was an important carbon source for microorganisms in yak rumen. Glycoside hydrolases (GHs) were the most abundant class of CAZymes, followed by glycosyl transferases (GTs), which were important to digestion of oil, cellulose, and hemicellulose in food. These results contribute to the understanding of microbial components and functions in yak rumen.

Genome-wide identification of SNPs associated with body weight in yak.

BACKGROUND: The yak is the most important livestock in the Qinghai-Tibet Plateau, and body weight directly affects the economic values of yak. Up to date, the genome-wide profiling of single-nucleotide polymorphisms (SNPs) associating with body weight has not been reported in yak. In the present study, the SNPs in 480 yaks from three breeds were analyzed using the commercial high-density (600 K) yak SNP chips. RESULTS: The results identified 12 and 4 SNPs potentially associated with body weight in male and female yaks, respectively. Among them, 9 and 2 SNPs showed significant difference in yak body weight between different genotypes at each locus in male and female yaks, respectively. Further exploration found 33 coding genes within the 100 kbp upstream or downstream to the SNP loci, which might be potentially affected by the variation of SNPs. Among them, G protein-coupled receptor kinase 4 (GRK4) might be potentially affected by the SNP AX-174555047, which has been reported to affect the functioning of two body-weight associated hormones (parathyroid hormone, PTH, and adrenomedullin, ADM). Determination of PTH and ADM levels in yak revealed positive relationship between PTH level and body weight, negative relationship between ADM level and body weight along with the variation of AX-174555047 mutation. CONCLUSIONS: These results suggested that the SNP AX-174555047 might potentially affect body weight through mediating GRK4 expression and then PTH and ADM functioning. Thus, the SNP AX-174555047 might be used as a biomarker for molecular breeding of yak. More investigations are required to validate this point.

Codon Usage Bias and Cluster Analysis of the MMP-2 and MMP-9 Genes in Seven Mammals.

Matrix metalloproteinase (MMP)-2 and MMP-9 are a family of Zn2+ and Ca2+-dependent gelatinase MMPs that regulate muscle development and disease treatment, and they are highly conservative during biological evolution. Despite increasing knowledge of MMP genes, their evolutionary mechanism for functional adaption remains unclear. Moreover, analysis of codon usage bias (CUB) is reliable to understand evolutionary associations. However, the distribution of CUB of MMP-2 and MMP-9 genes in mammals has not been revealed clearly. Multiple analytical software was used to study the genetic evolution, phylogeny, and codon usage pattern of these two genes in seven species of mammals. Results showed that the MMP-2 and MMP-9 genes have CUB. By comparing the content of synonymous codon bases amongst seven mammals, we found that MMP-2 and MMP-9 were low-expression genes in mammals with high codon conservation, and their third codon preferred the G/C base. RSCU analysis revealed that these two genes preferred codons encoding delicious amino acids. Analysing what factors influence CUB showed that the third base distributors of these two genes were C/A and C/T, and GC3S had a wide distribution range on the ENC plot reference curve under no selection or mutational pressure. Thus, mutational pressure is an important factor in CUB. This study revealed the usage characteristics of the MMP-2 and MMP-9 gene codons in different mammals and provided basic data for further study towards enhancing meat flavour, treating muscle disease, and optimizing codons.

Comparative transcriptome analysis of winter yaks in plateau and plain.

The yak is an important source for the people living and ecological environment in the Qinghai-Tibet Plateau. In every winter, many domestic yaks will lose bodyweight or dead under cold and food scarcity. Moving the plateau yaks to farm in the plain is a useful approach to reduce their environmental stress and gain more production. In this study, we measured growth, slaughter and beef quality traits every month and sequenced mRNA expression levels of muscles of two groups yaks living in plateau and plain respectively. We found there is significant difference (p-value <0.01) in the third (60 days), fourth (90 days), fifth (120 days) and sixth (150 days) weights between subpopulations in the plateau and plain. We identified 540 different expressed genes (DEGs) including 123 known genes and 417 unknown genes. Using the weighted correlation network analysis (WGCNA) to build a co-express network, the modules were strong relative to weight traits. The findings highlighted the underlying way and a relative network to yield a new view about gene expression between the yaks living plateau and plain.

Adipose-Secreted Exosomes and Their Pathophysiologic Effects on Skeletal Muscle.

Due to its prominent secretory activity, adipose tissue (AT) is now considered a major player in the crosstalk between organs, especially with skeletal muscle. In which, exosomes are effective carriers for the intercellular material transfer of a wide range of molecules that can influence a series of physiological and pathological processes in recipient cells. Considering their underlying roles, the regulatory mechanisms of adipose-secreted exosomes and their cellular crosstalk with skeletal muscle have received great attention in the field. In this review, we describe what is currently known of adipose-secreted exosomes, as well as their applications in skeletal muscle pathophysiology.

Differences in proteomic profiles between yak and three cattle strains provide insights into molecular mechanisms underlying high-altitude adaptation.

Yaks display unique properties of the lung and heart, enabling their adaptation to high-altitude environments, but the underlying molecular mechanisms are still largely unknown. In the present study, the proteome differences in lung and heart tissues were compared between yak (Bos grunniens) and three cattle strains (Bos taurus, Holstein, Sanjiang and Tibetan cattle) using the sequential window acquisition of all theoretical mass spectra/data-independent acquisition (SWATH/DIA) proteomic method. In total, 51,755 peptides and 7215 proteins were identified. In the lung tissue, there were 162, 310 and 118 differential abundance proteins (DAPs) in Tibetan, Holstein and Sanjiang cattle compared to yak respectively. In the heart tissue, there were 71, 57 and 78 DAPs in Tibetan, Holstein and Sanjiang cattle compared to yak respectively. The Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis revealed that the DAPs were enriched for the retinol metabolism and toll-like receptor categories in lung tissue. The changes in these two pathways may regulate hypoxia-induced factor and immune function in yaks. Moreover, DAPs in heart tissues were enriched for cardiac muscle contraction, Huntington's disease, chemical carcinogenesis and drug metabolism-cytochrome P450. Further exploration indicated that yaks may alter cardiac function through regulation of type 2 ryanodine receptor (RyR2) and Ca2+ -release channels. The present results are useful to further develop an understanding of the mechanisms underlying adaptation of animals to high-altitude conditions.

Whole-genome resequencing provides insights into the evolution and divergence of the native domestic yaks of the Qinghai-Tibet Plateau.

BACKGROUND: On the Qinghai-Tibet Plateau, known as the roof ridge of the world, the yak is a precious cattle species that has been indispensable to the human beings living in this high-altitude area. However, the origin of domestication, dispersal route, and the divergence of domestic yaks from different areas are poorly understood. RESULTS: Here, we resequenced the genome of 91 domestic yak individuals from 31 populations and 1 wild yaks throughout China. Using a population genomics approach, we observed considerable genetic variation. Phylogenetic analysis suggested that the earliest domestications of yak occurred in the south-eastern QTP, followed by dispersal to the west QTP and northeast to SiChuang, Gansu, and Qinghai by two routes. Interestingly, we also found potential associations between the distribution of some breeds and historical trade routes such as the Silk Road and Tang-Tibet Ancient Road. Selective analysis identified 11 genes showing differentiation between domesticated and wild yaks and the potentially positively selected genes in each group were identified and compared among domesticated groups. We also detected an unbalanced pattern of introgression among domestic yak, wild yak, and Tibetan cattle. CONCLUSIONS: Our research revealed population genetic evidence for three groups of domestic yaks. In addition to providing genomic evidence for the domestication history of yaks, we identified potential selected genes and introgression, which provide a theoretical basis and resources for the selective breeding of superior characters and high-quality yak.

Methylome and transcriptome profiles in three yak tissues revealed that DNA methylation and the transcription factor ZGPAT co-regulate milk production.

BACKGROUND: Domestic yaks play an indispensable role in sustaining the livelihood of Tibetans and other ethnic groups on the Qinghai-Tibetan Plateau (QTP), by providing milk and meat. They have evolved numerous physiological adaptations to high-altitude environment, including strong blood oxygen transportation capabilities and high metabolism. The roles of DNA methylation and gene expression in milk production and high-altitudes adaptation need further exploration. RESULTS: We performed genome-wide DNA methylome and transcriptome analyses of breast, lung, and biceps brachii muscle tissues from yaks of different ages. We identified 432,350 differentially methylated regions (DMRs) across the age groups within each tissue. The post-mature breast tissue had considerably more differentially methylated regions (155,957) than that from the three younger age groups. Hypomethylated genes with high expression levels might regulate milk production by influencing protein processing in the endoplasmic reticulum. According to weighted gene correlation network analysis, the "hub" gene ZGPAT was highly expressed in the post-mature breast tissue, indicating that it potentially regulates the transcription of 280 genes that influence protein synthesis, processing, and secretion. The tissue network analysis indicated that high expression of HIF1A regulates energy metabolism in the lung. CONCLUSIONS: This study provides a basis for understanding the epigenetic mechanisms underlying milk production in yaks, and the results offer insight to breeding programs aimed at improving milk production.

The whole-transcriptome landscape of muscle and adipose tissues reveals the ceRNA regulation network related to intramuscular fat deposition in yak.

BACKGROUND: The Intramuscular fat (IMF) content in meat products, which is positively correlated with meat quality, is an important trait considered by consumers. The regulation of IMF deposition is species specific. However, the IMF-deposition-related mRNA and non-coding RNA and their regulatory network in yak (Bos grunniens) remain unknown. High-throughput sequencing technology provides a powerful approach for analyzing the association between transcriptome-related differences and specific traits in animals. Thus, the whole transcriptomes of yak muscle and adipose tissues were screened and analyzed to elucidate the IMF deposition-related genes. The muscle tissues were used for IMF content measurements. RESULTS: Significant differences were observed between the 0.5- and 2.5-year-old yaks. Several mRNAs, miRNAs, lncRNAs and circRNAs were generally expressed in both muscle and adipose tissues. Between the 0.5- and 2.5-year-old yaks, 149 mRNAs, 62 miRNAs, 4 lncRNAs, and 223 circRNAs were differentially expressed in muscle tissue, and 72 mRNAs, 15 miRNAs, 9 lncRNAs, and 211 circRNAs were differentially expressed in adipose tissue. KEGG annotation revelved that these differentially expressed genes were related to pathways that maintain normal biological functions of muscle and adipose tissues. Moreover, 16 mRNAs, 5 miRNAs, 3 lncRNAs, and 5 circRNAs were co-differentially expressed in both types of tissue. We suspected that these co-differentially expressed genes were involved in IMF-deposition in the yak. Additionally, LPL, ACADL, SCD, and FASN, which were previously shown to be associated with the IMF content, were identified in the competing endogenous RNA (ceRNA) regulatory network that was constructed on the basis of the IMF deposition-related genes. Three ceRNA subnetworks also revealed that TCONS-00016416 and its target SIRT1 "talk" to each other through the same miR-381-y and miR-208 response elements, whereas TCONS-00061798 and its target PRKCA, and TCONS-00084092 and its target LPL "talk" to each other through miR-122-x and miR-499-y response elements, respectively. CONCLUSION: Taken together, our results reveal the potential mRNA and noncoding RNAs involved in IMF deposition in the yak, providing a useful resource for further research on IMF deposition in this animal species.

Signature of high altitude adaptation in the gluteus proteome of the yak.

Yak is the unique Bovidae species in the Qinghai-Tibetan Plateau. A previous proteomic study has compared the yak muscle tissue to one cattle strain using the isobaric tags for relative and absolute quantification approach. In this study, to further investigate the molecular mechanisms underlying yak adaptation, the proteomic profiles of gluteus were compared between yak and one moderate-altitude cattle strain (Tibetan cattle) and two low-altitude cattle strains (Holstein and Sanjiang cattle) using a label-free quantitative method. The comparisons identified 20, 364, 143 upregulated proteins and 4, 6, 37 downregulated proteins in yak, compared with Tibetan, Holstein, and Sanjiang cattle, respectively. Protein-protein interaction analysis indicated that these differentially expressed proteins were mainly related to "oxidative phosphorylation" and "electron transport chain." Further analysis revealed that NADH dehydrogenase [ubiquinone] 1 alpha subcomplex subunit 11, NADH dehydrogenase [ubiquinone] 1 beta subcomplex subunit 4, cytochrome C oxidase subunit 6A2, mitochondrial and cytochrome c oxidase subunit NDUFA4 were all increased in the yak, suggesting that yak might increase mitochondrial capacity to sustain metabolic rates under high altitude conditions, which might be a long-term adaptive mechanism underlying adaptation to high altitude environments. Yak increased the level of thioredoxin reductase 2 to protect themselves from oxidative damages. Moreover, the increased expression levels of phosphatidylinositol 4,5-bisphosphate 3-kinase catalytic subunit alpha isoform and caveolin-1 in yak suggested that yaks promoted glucose uptake for adaptation to high altitude. These results provided more information to better understand the molecular mechanisms underlying yak adaption.

Detection and integrated analysis of lncRNA and mRNA relevant to plateau adaptation of Yak.

Known as the 'ship of the plateau', through thousands of years evolution and cruelty environments selection containing low oxygen and strong ultraviolet radiation, yaks have adapted plateau environments and supplied important goods and materials for the people in the Qinghai-Tibet Plateau. This study aimed to identify differentially expressed (DE) genes and novel long non-coding RNAs (lncRNAs) of yaks for the Plateau adaptation and their underlying co-expression and regulatory network. We carried out RNA-seq analysis for cerebral and cerebellar tissue specimens of Bos taurus, Bos grunniens × Bos Taurus and B. grunniens. Furthermore, 12,072 pseudo lncRNAs were predicted using three software. In total, 4,257 significant DE transcripts were identified using the Ballgown R package (p < .01), of which 1,021 were protein-coding genes, 14 were known lncRNAs, and 661 were novel lncRNAs. Using WGCNA, a co-expression network of DE mRNAs and lncRNAs comprising 5 modules was generated to determine functional associations clusters. This study reveals a valuable sub-network comprising 8 hub genes, one known lncRNA and 5 novel lncRNAs in the major module. These hub genes are associated with blood pressure regulation, generation of reactive oxygen species and metabolism. The analysis of co-expressed genes thus provides a basis for the regulatory mechanisms in PA in Yaks and for the detection of additional genes between cross-breed and parent populations.

A global analysis of CNVs in diverse yak populations using whole-genome resequencing.

BACKGROUND: Genomic structural variation represents a source for genetic and phenotypic variation, which may be subject to selection during the environmental adaptation and population differentiation. Here, we described a genome-wide analysis of copy number variations (CNVs) in 16 populations of yak based on genome resequencing data and CNV-based cluster analyses of these populations. RESULTS: In total, we identified 51,461 CNV events and defined 3174 copy number variation regions (CNVRs) that covered 163.8 Mb (6.2%) of yak genome with more "loss" events than both "gain" and "both" events, and we confirmed 31 CNVRs in 36 selected yaks using quantitative PCR. Of the total 163.8 Mb CNVR coverage, a 10.8 Mb region of high-confidence CNVRs directly overlapped with the 52.9 Mb of segmental duplications, and we confirmed their uneven distributions across chromosomes. Furthermore, functional annotation indicated that the CNVR-harbored genes have a considerable variety of molecular functions, including immune response, glucose metabolism, and sensory perception. Notably, some of the identified CNVR-harbored genes associated with adaptation to hypoxia (e.g., DCC, MRPS28, GSTCD, MOGAT2, DEXI, CIITA, and SMYD1). Additionally, cluster analysis, based on either individuals or populations, showed that the CNV clustering was divided into two origins, indicating that some yak CNVs are likely to arisen independently in different populations and contribute to population difference. CONCLUSIONS: Collectively, the results of the present study advanced our understanding of CNV as an important type of genomic structural variation in yak, and provide a useful genomic resource to facilitate further research on yak evolution and breeding.

Transcriptome profile and unique genetic evolution of positively selected genes in yak lungs.

The yak (Bos grunniens), which is a unique bovine breed that is distributed mainly in the Qinghai-Tibetan Plateau, is considered a good model for studying plateau adaptability in mammals. The lungs are important functional organs that enable animals to adapt to their external environment. However, the genetic mechanism underlying the adaptability of yak lungs to harsh plateau environments remains unknown. To explore the unique evolutionary process and genetic mechanism of yak adaptation to plateau environments, we performed transcriptome sequencing of yak and cattle (Bos taurus) lungs using RNA-Seq technology and a subsequent comparison analysis to identify the positively selected genes in the yak. After deep sequencing, a normal transcriptome profile of yak lung that containing a total of 16,815 expressed genes was obtained, and the characteristics of yak lungs transcriptome was described by functional analysis. Furthermore, Ka/Ks comparison statistics result showed that 39 strong positively selected genes are identified from yak lungs. Further GO and KEGG analysis was conducted for the functional annotation of these genes. The results of this study provide valuable data for further explorations of the unique evolutionary process of high-altitude hypoxia adaptation in yaks in the Tibetan Plateau and the genetic mechanism at the molecular level.

An enzyme-free homogenous electrochemical assay for sensitive detection of the plasmid-mediated colistin resistance gene mcr-1.

Antibiotic resistance associated with the mcr-1 gene of Gram-negative bacteria, which confers resistance to drugs of last resort and has the potential to spread via plasmids, is one of the most pressing issues facing global health today. Point-of-care testing for the mcr-1 gene is needed to aid in the identification of colistin resistance in the field and to control its horizontal transmission. Here, we report the successful development of an enzyme-free homogenous electrochemical strategy for sensitive detection of the antibiotic resistance gene mcr-1 using the hybridization chain reaction and mcr-1-specific toehold probe. The long double-stranded DNA polymer produced using this strategy could be detected by assessing the diffusion of methylene blue towards the surface of a screen-printed gold electrode. Under optimized conditions, a linear relationship was observed between the variation of peak current and the natural logarithm of the mcr-1 gene concentration in the range of 1 nM to 1 µM with a detection limit of 0.78 nM (S/N = 3). This enzyme-free, isothermal platform is a rapid, portable, disposable, and sensitive method for detection of plasmid-mediated colistin resistance.

Optimization of the Original TRIzol-Based Technique Improves the Extraction of Circulating MicroRNA from Serum Samples.

BACKGROUND: MicroRNAs (miRNAs) are a group of small endogenous, non-coding RNA molecules that have been demonstrated to be essential regulators of many critical biological and pathological processes. Because of their high stability in blood and the strong implication of miRNA expression profiles for human diseases, miRNAs are currently emerging as promising circulating biomarkers for the diagnosis or prognosis of a variety of human diseases. The TRIzol-based technique has been widely used for cell or tissue RNA extraction because of its economy and reliability. However, the original TRIzol-based RNA isolation protocol was not specifically designed for microRNA extraction from serum samples. When it was used to extract serum microRNAs, due to the short sequence and low level of microRNAs, the isolation efficiency in most cases could not meet the requirement. To address this issue, in this study, an improved TRIzol-based protocol was established by modifying the extraction procedure of the original TRIzol-based protocol. The performance of the improved TRIzol-based protocol was evaluated by comparison with other methods. METHODS: Total RNA was isolated by the improved TRIzol-based method, the original method, and two other commonly used methods. RT-qPCR and spectrophotometry were used to examine the quality and yield of total RNA. RESULTS: The improved method was more efficient than the original protocol and more suitable for real-time PCR-based profiling experiments. CONCLUSIONS: The optimized TRIzol-based method described in this report is suitable for the extraction of serum microRNAs and useful for the development of microRNAs as diagnostic biomarkers.

Additional glial cell line-derived neurotrophic factor in vitro promotes the proliferation of undifferentiated spermatogonia from sterile cattleyak.

Cattleyak is a typically male sterile species. The meiosis process is blocked and the scarcity of spermatogenic stems cells are both contributing factors to the inability of male cattleyak to produce sperm. While Glial cell line-derived neurotrophic factor (GDNF) is the first discovered growth factor known to promote the proliferation and self-renewal of spermatogenic stem cells, its relationship to the spermatogenesis arrest of cattleyak remains unclear. In this report, we studied the differential expression of GDNF in the testis of yak and cattleyak, and discussed the optimal concentration of GDNF in the culture medium of undifferentiated spermatogonia (UDSPG) of cattleyak in vitro and the effect of GDNF on the proliferation of cattleyak UDSPG. The results indicated that GDNF expression in the testicular tissue of cattleyak was inferior to that of yak. Moreover, the optimum value for the UDSPG in vitro culture was determined to be 20-30 ng/mL for cattleyak. In vitro, the proliferation activity of UDSPG was observed to increase with additional GDNF due to the up-regulation of proliferation-related genes and the down-regulation of differentiation-related genes. We hereby report that the scarcity of cattleyak UDSPG is due to insufficient expression of GDNF, and that the addition of GDNF in vitro can promote the proliferation of cattleyak UDSPG by regulating the expression of genes related to proliferation and differentiation. This work provides a new insight to solve the issue of spermatogenic arrest in cattleyak.