Transcriptomics in atopic dermatitis / 中华皮肤科杂志

With the development of transcriptomic technologies such as gene chip technology and RNA sequencing technology, important related factors in the pathogenesis of atopic dermatitis (AD) have been gradually identified, such as different T helper (Th) cell subtypes and other immune-related cells (macrophages and Langerhans cells) ; abnormal changes in active substances such as interleukin-4, interleukin-13, fillagrin and loricrin released by immune-related cells such as Th2 cells and keratinocytes have been found to play major roles in pruritus and skin barrier damage in AD. In recent years, transcriptomic technologies have been applied to the analysis of changes in transcriptomic profiles of patients before and after treatment to evaluate patients′ condition and therapeutic effect. This review summarizes research progress in transcriptomics in AD in recent years.

Expression profile and clinical significance of miRNA in children with autism spectrum disorder / 中国医师杂志

Objective:The purpose of this study was to analyze the expression profile of miRNAs in children with autism spectrum disorders (ASD), and to discuss the clinical significance of differentially expressed miRNAs.Methods:MiRNA microarray was used to analyze the expression of miRNA in peripheral blood of 3 pairs of ASD patients-healthy controls; 17 pairs of ASD patients-healthy controls were used to verify the differentially expressed miRNA; Receiver operating characteristic (ROC) curve was used to analyze the differential expression the value of miRNA in the diagnosis of ASD.Results:A total of 32 differentially expressed genes were screened by 3 pairs of miRNA microarray including 12 up-regulated miRNAs and 20 down-regulated miRNAs. miRNA verification of 20 differentially expressed miRNAs showed miR-15a-5p, miR-27a-3p , miR-142-3p and miR-142-5p were significantly down-regulated in children with ASD, with statistically significant difference (all P<0.05). ROC curve analysis showed that the area under the curve (AUC) of the above four miRNAs diagnosing ASD were all greater than 0.70, with sensitivities 94.12%, 100%, 100%, and 82.35%, respectively. Conclusions:The expression of miR-142-3p, miR-27a-3p/miR-15a-5p, and miR-142-5p is down-regulated in the peripheral blood of ASD patients, and has the potential as biomarkers for early screening of ASD.

Screening of DNA methylation sites associated with melanoma and construction of an aberrant methylation profile: a preliminary study / 中华皮肤科杂志

Objective:To screen aberrant DNA methylation sites associated with melanoma using gene chip technology, and to preliminarily construct a melanoma-specific methylation profile.Methods:The Illumina Human Methylation 450K whole-genome methylation chip was used to detect the whole-genome DNA in 6 melanoma tissues and their paralesional skin tissues, and DNA differentially methylated sites were obtained. Gene Ontology (GO) enrichment analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) -based pathway analysis were carried out to investigate gene functions.Results:Gene chip testing showed that there were 27 779 differentially methylated sites between melanoma tissues and paralesional tissues, of which 16 673 were hypermethylated sites and 11 106 were hypomethylated sites in melanoma tissues. According to more stringent screening criteria " P < 0.01 and |Δβ| > 0.2", a total of 4 883 differentially methylated sites were screened out after filtering out all single nucleotide polymorphism-related probes, probes located on the XY chromosomes and cross-reactive probes, 1 459 (30%) of which were located in the promoter region including TSS1500, TSS200, 5′UTR and 1st Exon. GO enrichment analysis showed that differentially methylated genes were involved in many biological processes, including cell growth, differentiation, adhesion, movement and migration, signal transduction, transcriptional regulation, etc. KEGG-based pathway analysis showed that differentially methylated genes were mainly involved in signaling pathways, such as focal adhesion pathway, cancer pathways, transforming growth factor-β signaling pathway, phosphatidylinositol signaling pathway, melanogenesis pathway, chemokine signaling pathway, adhesion junction pathway, calcium signaling pathway, cell adhesion molecule pathway, mitogen-activated protein kinase signaling pathway, Wnt signaling pathway, Janus kinase-signal transducer and activator of transcription signaling pathway. Based on the criteira "the top 16 most differentially methylated genes related to hypermethylated sites in the promoter region, the genes with the highest methylation frequency (CpG sites ≥ 7) , the genes with certain functions or involved in a certain signaling pathway", 8 genes (KAAG1, DGKE, SOCS2, TFAP2A, GNMT, GALNT3, ANK2 and HOXA9) were selected as candidate biomarkers for melanoma. Conclusion:There are many hypermethylated genes in melanoma tissues, and 8 differentially methylated genes may serve as biomarkers for melanoma.

Estimating Postmortem Interval by Protein Chip Detection Technology Combined with Multidimensional Statistics / 法医学杂志

Objective To obtain the protein expression profile of rat liver tissue after death by the 2100 bioanalyzer combined with protein chip, and infer the relationship between protein expression profile and postmortem interval. Methods Rats were killed by abdominal anesthesia and placed at 16 ℃. Water-soluble proteins in liver tissues were extracted at 14 time points after death. The expression profile data of proteins with relative molecular weight of 14 000-230 000 were obtained using protein chip, and principal component analysis （PCA）, partial least squares-discriminant analysis （PLS-DA） and Fisher discriminant were used to analyze the data. Results According to the changes of protein expression profile, the postmortem interval was divided into group A （0 d）, group B （1-9 d）, group C （12-30 d） according to the result of PLS-DA. The prediction accuracy of the training set and test set of the model were all 100.0%, and the internal cross-validation of the training set was 100.0% according to Fisher discriminant. The Fisher discriminant model at each time point of group B and C was established to narrow the time window of postmortem interval estimation. The prediction accuracy of the training set and test set were all 100.0%, and the internal cross-validation accuracy of the training set was 100.0% in group B. The prediction accuracy of the training set and test set were respectively 95.2% and 78.6% in group C, and the internal cross-validation of the training set was 88.1%. Conclusion Protein chip detection technology can quickly and easily obtain the expression profile of water-soluble proteins of rat liver tissue with a relative molecular weight of 14 000-230 000 at different time points after death. PLS-DA and Fisher discriminant models are established to classify and predict the postmortem interval, in order to provide new ideas and methods for postmortem interval estimation.

Bone morphogenetic protein-9 regulates expression of long non-coding RNAs in breast cancer cells / 肿瘤

Objective: To screen out the long non-coding RNAs (LncRNAs) related to bone morphogenetic protein-9 (BMP-9) expression, and to investigate the role of BMP-9 in the growth, differentiation, migration and apoptosis of breast cancer cells by regulating the expression of LncRNA. Methods: MDA-MB-231 cells were infected with the recombinant adenovirus Ad-BMP-9 carrying whole BMP -9 gene (named as MDA-MB-231/BMP-9 cells) or the empty vehicle adenovirus Ad-GFP carrying green fluorescent protein (GFP) gene (named as MDA-MB-231/GFP cells), respectively. Microarray technology was used to detect the difference in LncRNAs expression between MDAMB- 231/BMP-9 and MDA-MB-231/GFP cells. The changes of LncRNA LINC00443, LINC00638, LINC00486, RHNO1, SERHL, HOXA11-AS, IQCA1, LINC00461, LOC440173, LHFPL, ANKRD36BP2, BVES-AS1, LINC00937 and LINC00608 were validated by real-time fluorescent quantitative PCR. The biological funcation and related pathways of the above LncRNAs were analyzed by gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) databases. Results: The expression level of BMP-9 mRNA in MDA-MB-231/BMP-9 cells was significantly higher than that in MDA-MB-231/GFP cells (as the control). Compared with the control group, the expression levels of LncRNAs in MDA-MB-231/BMP-9 cells changed significantly. The expression levels of LINC00443, LINC00638 and LINC00486 were up-regulated, while the expression levels of IQCA1, LINC00461, LOC440173, LHFPL and ANKRD36BP2 were down-regulated. The altered LncRNAs participated in the formation of cytoskeleton, cell membrane and so on, or played roles as signal molecules in intercellular signal transduction. Conclusion: The expression profile of LncRNAs in MDA-MB-231 cells with BMP-9 overexpression is significantly changed, indicating that LncRNAs may play key roles in the proliferation and invasion of breast cancer MDA-MB-231 cells regulated by BMP-9.

Correlation of metallothionein-2A, E-cadherin and cyclin E with biochemical recurrence in prostate cancer / 肿瘤研究与临床

Objective To investigate the expressions of metallothionein-2A (MT-2A), E-cadherin, interleukin-6 (IL-6), cyclin E, proliferating cell nuclear antigen (PCNA) and bcl-2 in prostate cancer tissues and their correlation with biochemical recurrence of prostate cancer. Methods Tissue specimens from 128 cases of prostate cancer who underwent radical prostatectomy in Shanxi Dayi Hospital from October 2012 to October 2017 were processed and transferred into tissue microarrays, the clinicopathological parameters of patients were also recorded. The expression levels of MT-2A, E-cadherin, IL-6, cyclin E, PCNA and bcl-2 were detected by immunohistochemical avidin-biotin complex (ABC) staining. The correlation between different molecular markers and biochemical recurrence of prostate cancer was analyzed. Results The biochemical recurrence rate of 128 patients with prostate cancer was 30.5％ (39/128). The biochemical recurrence rates of low-risk, intermediate-risk and high-risk prostate cancer patients were 14.8％(8/54), 38.7％(24/62) and 58.3％ (7/12), respectively. The risk classification and pathological T stage of patients with prostate cancer were associated with the expressions of MT-2A, cyclin E, IL-6 and E-cadherin (all P< 0.05). Multivariate Cox risk model showed that the high risk classification (HR= 1.81, 95％CI 1.56-2.19, P=0.042), MT-2A positive expression (HR= 2.01, 95％CI 1.08-3.15, P= 0.005), cyclin E positive expression (HR= 1.79, 95％CI 1.08-2.21, P= 0.042) and E-cadherin negative expression (HR= 1.92, 95％ CI 1.22-2.45, P= 0.020) were the independent risk factors for biochemical recurrence of prostate cancer. Conclusion The expression of MT-2A, cyclin E and E-cadherin may serve as independent predictors for biochemical recurrence of prostate cancer.

POCT in diagnosis of infectious diseases—the state of art and future / 中华检验医学杂志

The pathogen diagnosis of infectious diseases plays a critical role in the rational application of antibiotics , the monitoring and management of disease epidemiology and the control of drug -resistant bacteria in hospitals and communities .The ideal method for rapid detection of infectious diseases should be sensitive, rapid, specific, stable, low-cost and easy to use.This article reviews the clinical application of POCT in infectious disease antigen detection , antibody detection , nucleic acid detection , etc. and focuses on the advantages and potential of microfluidic technology in pathogen multiplex nucleic acid detection and drug resistance detection .Choosing the right time , the right place , and the right on-site rapid diagnostic technology will provide a new strategy for the diagnosis and treatment of infectious diseases in the graded diagnosis and treatment mode .

The application of microfluidic systems in clinical diagnostics / 中华检验医学杂志

Currently, clinical laboratories are developing towards two different directions .One is centralized laboratory with automated pipelines , and the other is highly integrated portable detection system.The latter evolved from an early single test device gradually to an integrated platform based on microfluidic technology , which can operate multiple steps and tests simultaneously .The microfluidic system is further developed to a miniaturized device which can perform biochemical , immune, microbial and cytological tests.With the medical reform process in China , the microfluidic systems will appear in the primary health unit or even families .This review concludes the current application of microfluidic technology in clinical diagnostics and its future trends .

Screening of Differential DNA Methylation Loci in Colorectal Cancer by Gene Microarray Technique / 胃肠病学

Background:DNA methylation is a vital part of epigenetic modification,and is closely related with the development and progress of multiple tumors such as colorectal cancer. However,its mechanism is not fully clarified. Screening specific methylation gene and construct the methylation expression profile of tumor has become the current research hotspot. Aims:To screen the differential methylation loci in colorectal cancer and para-cancerous normal tissue by gene methylation microarray technique,and to construct specific differential methylation gene profile of colorectal cancer. Methods:Methylation 450K bead-chip was applied to detect the methylation status in colorectal cancer and para-cancerous normal tissues of 6 cases. A total of 431 467 loci were analyzed and compared. Aberrant methylation loci were screened according to P value,and the hypermethylation loci and hypomethylation loci were differentiated by delta beta value. Moreover,the function of differential methylation gene was further analyzed by GO analysis and KEGG analysis. Results:A total of 3 649 differential methylation loci were found by comparing colorectal cancer tissue and para-cancerous normal tissue,including 1 259 hypermethylation loci,which mainly located in promoter and genosome,and 2 390 hypomethylation loci,which mainly located in intergenic region and genosome. A panel of aberrant methylation gene loci was screened out,including hypermethylation gene loci such as SLC15A3 and hypomethylation gene loci such as ACOT2,TTLL8 and UHRF1. GO analysis and KEGG analysis showed that the function of these genes might be correlated with DNA binding,transcription factor activity and signal transduction pathway. Conclusions:There are many differential methylation loci in colorectal cancer and para-cancerous normal tissues,suggesting that aberrant DNA methylation is closely related with the development and progress of colorectal cancer. DNA methylation microarray technique could be applied for preliminary screening of differential methylation loci. However,constructing the differential methylation profile in colorectal cancer as a clinical biomarker should be further validated.

Identification and analysis of exosomal miRNAs in human umbilical vein endothelial cells irradiated by 60 Co γ-rays / 军事医学

Objective To study the changes in miRNAs expression in the exosomes of human umbilical vein endothelial cells(HUVECs) after 60 Co γ-rays expose using microRNA(miRNA) chips and bioinformatics techniques so as to provide new clues to the mechanism of radiation-induced vascular tissue injury and its bystander effects.Methods HUVECs exosomes were collected in the control and 4 Gy irradiated cells by ultra-high-speed centrifugation,and further confirmed using transmission electron microscopy (TEM) and Western blotting of exosomes biomarkers.miRNA microarray was used to analyze miRNA expression profiles of exosomes and cells.Also,real-time quantitative PCR(qRT-PCR) was used to verify differentially expressed miRNAs,and the miRDB and TargetScan were performed to predict the target genes of the differentially expressed miRNAs.Bioinformatics analysis was performed using DAVID,KEGG and other online tools.Results Compared with the control exosomes from non-irradiated HUVECs,miRNA microarray analysis revealed that 5 up-regulated,and 13 down-regulated miRNAs were identified in the exosomes from HUVECs at 0.5 h after 4 Gy-irradiation,and 16 up-regulated and 5 down-regulated miRNAs at 2 h after 4 Gy-irradiation.Moreover,38 and 85 miRNAs were differentially expressed respectively in the HUVECs at 0.5 h and 2 h after radiation.The difference was statistically significant(P<0.01).The results of bioinformatics showed that these miRNAs might exert the radiation-induced bystander effect (RIBE) by regulating MAPK signal pathways,RAS and PI3K-Akt signal pathways.Conclusion The ionizing radiation injury significantly alters the components and expression levels of exosomal miRNAs,which play important roles in regulating the signal pathways in response to radiation.

Expression and bioinformatics analysis of long-chain non-coding RNA PVT1 in tumors / 中华病理学杂志

Objective@#To study the expression and mechanism of long-chain non-coding RNA PVT1 in tumor by bioinformatics analysis and experimental verification, and to provide new ideas for the study of the pathogenesis of tumors.@*Methods@#The expression of PVT1 in 14 common tumors was downloaded from starBase v2.0 public database, which also was verified by PVT1 RNA-in situ hybridization.The upstream transcription factors, the downstream target microRNA(miRNA) for PVT1 and the target genes for the target miRNAs were predicted and analyzed by using bioinformatics based on the database of UCSC Genome Browser, HMDD v2.0, miRTar Base, JASPAR databases.@*Results@#StarBase database analysis and RNA in situ hybridization showed that PVT1 was highly expressed in kidney clear cell carcinoma and colon and rectal adenocarcinoma. PVT1 was regulated by the upstream transcription factors CREB1, Atf1, SP1, KLF5, STAT3, while it could control the expression of the downstream target miR-16. bcl-2, VEGFA, CCNE1, CCND1 and SHOC2 showed an interaction with the transcription factor of PVT1, which formed a feedback regulatory pathway.@*Conclusions@#PVT1 is highly expressed in kidney clear cell carcinoma and colon and rectal adenocarcinoma.The predictive analysis of bioinformatics demonstrates that transcription factor/PVT1/miR-16/target gene signal axon may be an important molecular mechanism, which provide a valuable clue for further functional mechanism research of long-chain non-coding RNA.

LncRNAs expression profile of ankylosing sporidylitis revealed by microarray / 中国医师杂志

Objective To investigate the expression profile variation of long non-coding RNAs (lncRNAs) in ankylosing sporidylitis (AS) and explore the role of lncRNAs in the pathogenesis of AS.Methods The peripheral blood mononuclear cells of AS patients and health controls (HC) were used to detect for differently expressed lncRNAs by microarray.The roles of lncRNAs were predicted with GO and pathway analysis.The results were verified by real time-polymerase chain reaction (PCR).Results A total of 148 lncRNAs and 134 mRNAs were detected,which had more than 2-fold differentially expressed in AS patients.Bioinformatics analysis found that GO term enrichment included protein binding,regulation of transcription,metabolism,signal transduction,et al.and might involve in toll-like receptor pathway,protein kinase,complement pathway,notch signaling pathway and so on.The expressions of three lncRNAs were estimated by real time-PCR which found that consistent with that of microarrays.Among these,D90064 was the most aberrantly expressed lncRNAs.Conclusions Several lncRNAs expression was changed significantly in AS patients in comparison with HC,which implies that those different lncRNAs may have an important role in the development and progression of AS.

MicroRNA expression profiles in peripheral blood and skin lesions of patients with psoriasis vulgaris / 中华皮肤科杂志

Objective To investigate microRNA(miRNA)expression profiles in peripheral blood and skin lesions of patients with psoriasis vulgaris(PV), and to seek miRNAs consistently expressed in peripheral blood and skin lesions. Methods A miRNA microarray was used to screen differentially expressed miRNAs in skin lesions and peripheral blood samples between 17 patients with PV and 4 healthy human controls, and real?time fluorescence?based quantitative PCR(RT?qPCR)to verify the differentially expressed miRNAs. The correlations of these differentially expressed miRNAs with psoriasis area and severity index(PASI)score were assessed by Pearson correlation analysis. Results The Agilent human miRNA microarray profiling revealed 15 miRNAs consistently expressed in skin lesions and peripheral blood of patients with PV. Of the 15 miRNAs, 7 were verified as consistently expressed miRNAs by RT?qPCR. Among the 7 miRNAs, the expression intensity of miR?30e?5p, miR?192?5p, miR?17?3p and miR?1227?5p was negatively correlated with PASI scores(all P0.05). Conclusion Some miRNAs are consistently expressed in skin lesions and plasma of PV patients, which are expected to serve as biomarkers for evaluation of psoriasis severity.

MicroRNA expression profiles in early mycosis fungoides / 中华皮肤科杂志

Objective To screen microRNAs(miRNAs)related to early mycosis fungoides(MF). Methods A high?throughput miRNA PCR array was used to determine miRNA expression profiles in skin lesions of 6 patients with early MF (early MF group) and 6 patients with lichen planus (control group), followed by screening of differentially expressed miRNAs between the two groups. Then, real?time fluorescence?based quantitative PCR(RT?qPCR)was performed to verify the differentially expressed miRNAs in lesional specimens from 13 patients with early MF and 13 patients with eczema or lichen planus, as well as in Myla cells and normal human T?lymphocytes. Results The high?throughput miRNA PCR array showed that the expressions of hsa?miR?378a?5p, hsa?miR?107 and hsa?miR?302c?3p were significantly higher in the early MF group than in the control group(all P<0.05). For skin lesions, the results from RT?qPCR were similar to those from the miRNA array assay. Compared with normal human peripheral blood T?lymphocytes, Myla cells showed significantly increased expressions of hsa?miR?378a?5p and hsa?miR?107, which was consistent with the results from the miRNA array assay. However, no significant difference was observed in the expression of hsa?miR?302c?3p between the two kinds of cells. Conclusion MiRNA expression profiles in early MF are different from those in inflammatory skin diseases.

Array profiling identified MiRNAs dysregulation in laryngeal squamous cell carcinoma / 中国耳鼻咽喉头颈外科

OBJECTIVE The purpose of this study was to analyze the screened miRNAs related to tumorigenesis using miRNA array in laryngeal squamous cell carcinoma (LSCC), and provide a set of miRNAs that may be useful for the development of novel diagnostic markers and/or more effective therapeutic strategies from the screened miRNAs in LSCC. METHODS A total number of 5 patients who underwent surgery for primary laryngeal squamous cell carcinoma were recruited for miRNA array analysis. LSCC tissues compared with corresponding adjacent non-neoplastic tissues were analyzed by the Affymetrix GeneChip miRNA Array 3.0 to screen effective miRNAs, and the raw dataset had been submitted to Gene Expression Omnibus. Then mirfocus 3.0 database was adopted to analyze putative regulated miRNAs related to MCM4, a gene related to tumorigenesis we had studied previously in LSCC. Moreover, the selected putative regulated miRNAs were also validated by qRT-PCR in another 21 patients diagnosed as LSCC. RESULTS Analyzed by the miRNAs arrays, there were 127 miRNAs significantly related to tumorigenesis, and 78 showed a higher expression in tumor than in non-tumor tissue while 49 presented the contrasting pattern (P<0.01). Then analyzed by mirfocus 3.0 database, there were 2 putative regulated miRNAs, hsa-miR-24-3p and hsa-miR-183-5p, related to the expression of MCM4. Another miRNA we should focus on was hsa-miR-30a-5p, which was down-expressed obviously analyzed by the miRNA array. The expression of the 3 putative regulated miRNAs were also validated by qRT-PCR in another 21 patients, and the result was the same with that in miRNA array (P<0.05). CONCLUSION The 3 putative miRNAs based on miRNA array analysis, hsa-miR-24-3p, hsa-miR-183-5p and hsa-miR-30a-5p, could be considered as potential diagnostic and therapeutic markers in LSCC. The result will contribute to the understanding of the molecular basis of LSCC and help to improve the treatment.

Studies on the long non-coding RNA during the reprogramming of human pluripotent stem cells / 天津医药

Objective To investigate the changes and roles of the long non-coding RNA (IncRNA)during the reprogramming of human induced pluripotent stem cells. Methods Agilent Human lncRNA (4 × 180K) chip was used to check the expression of lncRNA in somatic cells, induced pluripotent stem cells and embryonic stem cells. Compared with differentially expressed lncRNA in somatic cells and induced pluripotent stem cells, lncRNA was selected that may play an important role during the reprogramming of human pluripotent stem cells. Results The lncRNA expression profiles in induced pluripotent stem cells were similar to embryonic stem cells, but were different from the somatic cells. A total of 3 156 differentially expressed lncRNAs were found between stem cells and somatic cells by cluster analysis, and 222 differentially expressed lncRNAs were found during the reprogramming process of human pluripotent stem cells by biological analysis. Conclusion lncRNA may play an important role in reprogramming process of human pluripotent stem.

Activation of Wnt signaling pathway in the neuronal differentiation of bone marrow mesenchymal stem cells：a microarray analysis / 中国组织工程研究

BACKGROUND:It wil provide a new insight into the future application of bone marrow mesenchymal stem cels in the treatment of spinal cord injury and tissue engineering by studying the effect of activation of Wnt signaling pathway in the neuronal differentiation of bone marrow mesenchymal stem cels. OBJECTIVE: To detect the expression of related genes by gene chip technology during the neuronal differentiation of bone marrow mesenchymal stem cels. METHODS:Human bone marrow mesenchymal stem cels were isolated and purified, and passage 5 cels were obtained. GatewayTM technology was used to build lentiviral vectors that was used to transfect Wnt-1 into human bone marrow mesenchymal stem cels. Control, non-transduction and transduction groups were set in this study. Human bone marrow mesenchymal stem cels were then induced to differentiate into neurons. Cel morphology was observed under inverted phase contrast microscope. Gene chip was used to detect the regulation changes and the differential expression of related genes in the Wnt signaling pathway. RESULTS AND CONCLUSION: Under the scanning electron microscope, the transfected cels were found to have the similar morphology of neuron-like cels. Analysis by the gene chip hybridization technique showed that 3 287 genes were up-regulated and 4 215 genes were down-regulated in the signal pathway. In the Wnt signaling pathway, genes related to the nervous system development and differentiation were up- or down-regulated. It is verified that the Wnt signal pathway is activated via Wnt-1 transduction, and the downstream genes appear to have genetic transcription so as to promote the neuronal differentiation of human bone marrow mesenchymal stem cels.

Detecting JAK2 V617F mutation in myeloproliferative neoplasms by microarray based digital PCR / 中华检验医学杂志

Objective To evaluate the sensitivity, repeatability and accuracy of microarray digital PCR system in detecting JAK2 V617F mutation, which was closely related to myeloproliferative neoplasms (MPN).Methods All of the 31 MPN patients with JAK2 V617F mutation, including 18 cases of polycythemia vera(PVs),11 primary thrombocythemias (ETs) and 2 primary myelofibrosis (PMFs), were collected from Huashan Hospital, Fudan University during 2014 -2015, while 10 normal controls and 6 cases with abnormal increased hemoglobin were involved.Human erythroleukemia cell line ( HEL ) and colorectal cancer cell SW480 were used as the mutant and the wild type control, respectively.The sensitivity of microarray digital PCR were verified by detecting the gradient diluted mutation standard harboring 30%, 10%, 1%, 0.1%and 0.01%mutant allele burden, respectively .Repeatability was evaluated by detecting 1%and 10% mutated samples for 5 times, respectively.MGB probe real time PCR was selected as the reference method to verify the accuracy of the digital PCR.Results With digital PCR, the accurate quantitation of JAK2 V617F mutation was achieved down to 0.1%, which is approximate to 0.16 copies per microliter.The results obtained from the two kinds of technique showed a high correlation by linear regression analysis (R2 =0.998 3).The results of repeated samples showed CVs as 17.18% for 1%mutant allele burden and 7.50%for 10%.Among all cases, the 31 patients known mutated were detected as positive and 10 controls as negative by both digital PCR and Real time PCR.In another 6 cases, 2 were found JAK2 V617F mutation of low allele burdens of 0.37% and 0.18% by digital PCR but detected as negative by real time PCR.Conclusions Microarray digital PCR offers a higher sensitivity and better repeatability than real time PCR which could help detect rare JAK2 V617F mutations in MPNs accurately.

Gene chip technique in detection of vancomycin-resistant Enterococcus gene / 军事医学

Objective To develop a detection method based on the technology of gene chips which can quickly distinguish genes of Enterococcus faecalis, E.faecium and vancomycin resistance.Methods Based on the specific gene ( ddl) sequences of two types of Enterococcus from GenBank, oligonucleotide probes which could detect and distinguish special genes and drug resistance genes ( vanA,vanB) of Enterococcus were designed and compounded.Then,the probes were dotted to modified slide.The target DNA fragments of vancomycin-resistant Enterococcus ( VRE) were labeled with biotin by multiple PCR amplification, and then hybridized with oligonucleotide probes on slide.The results were analyzed by portable imager.The multiple PCR system, hybridization reaction and condition of the chemiluminescence method were optimized before the specificity, sensitivity and reproducibility of the chip were evaluated.Results One universal primer, four specific primers, one universal probe and four specific probes were selected.This gene chip was demonstrated of high specificity and repeatability.The detection sensitivity was 103 CFU/ml.The gene chip detection results of 10 clinical samples were basically consistent with the drug sensitivity test ( 8/10 ) .Conclusion A gene chip technique for the detection of VRE is established successfully.It is possible to distinguish the type of VRE and detect the genetic phenotypes of drug resistance by gene chip technique.

Analysis and verification of gastric cancer metastatic lymphonodus associated long non-coding RNA / 中华普通外科杂志

Objective To analyze and verify the expression profiles of long non-coding RNAs (lncRNAs) in gastric cancer (GC) metastatic lymphonodus.Methods Microarray analysis was performed in 3 GC positive lymphonodus and 1 normal lymph node with Agilent Array platform to measure the expression levels of lncRNAs and mRNAs and to investigate the expression differences of lncRNAs in GC metastatic lymphonodus and normal lymphonodus, and hierarchical clustering used to screen out the differently expressed lncRNAs.15 up-regulated lncRNAs and 15 down-regulated lncRNAs were randomly chosen and RT-PCR was used to verify the expression differences.Results Comparing with normal lymphonodus, 353 lncRNAs and 547 mRNAs are up-regulated, but 464 lncRNAs and 562 mRNAs are down-regulated in GC metastatic lymphanodus as 6 times or more variation was found.The expressions of lncRNA OR3A4, LOC84740, FCGR1C and C21orf 96 were increased in GC metastatic lymphonodus, but lncRNA MSTO2P, LOC344595, TUG1, TYW3 and KRT8P10 decreased.Conclusions LncRNAs are aberrantly expressed in GC metastatic lymphonodus.