Establishment and verification of a real-time quantitative PCR method for determination of exogenous antibody gene copy number in CHO cells / 中国生物制品学杂志

@#Objective To establish a real-time quantitative PCR method using SYBR GreenⅠto detect the copy numbers of light chain(LC)and heavy chain(HC)of exogenous antibody gene in CHO cells,and verify and preliminarily apply this method.Methods With the B2m(β2-microglobulin)expressed stably in CHO cells as the internal reference gene,suitable primers of LC,HC genes and internal reference gene were designed respectively,and the reaction system and program of the real-time quantitative PCR method were determined. The established method was verified for the specificity,linearity,precision and durability,and used to detect the copy numbers of LC and HC genes in the recombinant cell lines of working cell bank(WCB)and cells of different passages.Results The primers of exogenous genes and internal reference gene showed specific binding to the target fragments;The efficiency of primer amplification for the B2m gene,LC gene,and HC gene was 106. 7%,106. 3% and 99. 1%,respectively,and the correlation coefficients of the linear equations were all greater than 0. 99 with a good linear relationship;The relative standard deviations(RSDs)of precision verification were all less than 1%;Few cycles of freeze-thaw in a short period had little effect on the detection results. The copy numbers of LC and HC genes in different generations of recombinant cell lines detected by the established method showed no obvious changes.Conclusion A real-time quantitative PCR method for the determination of the copy number of exogenous genes in CHO cells was successfully established with good specificity,linearity,precision and durability,which provides a reference for detecting the copy number of exogenous genes expressed in other CHO cell lines

Detecting the copy number of orfA gene to identify Brucella species and biovars by a real-time quantitative PCR assay / 中华地方病学杂志

Objective:To establish a real-time quantitative PCR assay to detect the copy number of IS711 transposase gene (orfA) in Brucella genome, and the assay is applied to identify the species and biovars of Brucella. Methods:To establish an orfA gene copy number detection system based on Taqman real-time quantitative PCR technique. Primers and probes of bcsp31 and orfA genes were designed, the contents of bcsp31 gene and orfA gene in the same strain with the same DNA concentration were simultaneously detected by real-time quantitative PCR assay, and cycle number (CT value) of the two genes were obtained. According to the differences of CT values of bcsp31 gene and orfA gene, the copy number of orfA gene in Brucella genome was calculated. At the same time, the DNA of Brucella 16M strain was double decreasing dilution to verify the stability of the detection system. Results:A real-time quantitative PCR assay was used to detect bcsp31 gene and orfA gene simultaneously, when the DNA concentration difference of 16M strain was 2 times, the mean difference of CT values measured was 1.00, 95% confidence interval was 0.95-1.05, standard deviation was 0.17, and coefficient of variation was 0.17. The orfA gene copy number of 30 Brucella strains was detected by this detection system. It was found that there were 6, 9, and 7 copy numbers in the biovars 1-3 of Brucella melitensis, respectively. The strain of Brucella suis biovar 2 had 10 copy numbers, which were different from those of the other 4 strains of biovars 1, 3-5. There were 37 copy numbers in Brucella ovis strain. The copy numbers were stable at 5-6 copies in 8 biovars (1-7, 9) of Brucella abortus strains. Conclusions:A real-time quantitative PCR assay for detection of orfA gene copy number in Brucella DNA has been established. This method could identify some Brucella species and biovars strains.

Establishment of qRT-PCR for absolute quantitative detection of Chikungunya virus / 中国热带医学

@#Abstract: Objective　To develop a real-time fluorescent quantitative RT-PCR (qRT-PCR) method for qualitative and quantitative Chikungunya virus (CHIKV) analysis. Methods Based on the systematic analysis of the genomic sequences of Chikungunya and its related arboviruses, the specific nucleic acid sequences for Chikungunya virus were screened and identified, and then the primers and TaqMan probe were designed. Meanwhile, the human GAPDH gene was used as an internal reference. The reaction system for qRT-PCR was systematically optimized by L9(34) orthogonal design, and a rapid detection method for Chikungunya by qRT-PCR based on TaqMan probe methods was established. The sensitivity, specificity, reproducibility, and coverage of the established method were analyzed in detail. The standard curve was made, and the absolute quantitative method was established using the cloned nucleic acid fragments as positive samples. Results　A real-time fluorescent quantitative RT-PCR assay was developed for the qualitative and quantitative analysis of Chikungunya virus. The reaction system included Chikungunya virus and reference internal gene specific primers and probe, RT/Taq enzyme mixture, reaction buffer, and negative and positive reference. The established method obtained positive results with the ROSS strain of ECSA subtype, LR2006 strain of IOL branch, 181/25 strain of Asian type and Dongguan 2010 epidemic strains of Chikungunya virus, but there was no cross-reaction with other 18 arboviruses belonging to Flaviviruses, Alphaviruses and Bunyavirus. The minimum detection limit of the established method was 5.80 copies/mL, and a linear relationship was observed between the amount of input plasmid DNA and fluorescence signal value over a range of 5.80×102 copies/mL to 5.80×1010 copies/mL, and the correlation coefficient was 0.999 5. The qRT-PCR amplification efficiency was 91%, and the intra-assay variations and inter-assay variations were 0.01-0.07 and 0.03-0.11, respectively. Conclusions　The TaqMan qRT-PCR method developed in this study can qualitatively and quantitatively detect Chikungunya virus rapidly with specificity and sensitivity, providing a technical method for the prevention and control of this viral disease.

Correlation of quantitative polymerase chain reaction with clinical characteristics of patients with viral retinitis

Purpose: To evaluate the correlation of quantitative real?time polymerase chain reaction (qRT?PCR) to the clinical characteristics of patients with viral retinitis.Methods: Retrospective case series. Results: Aqueous or vitreous samples of 20 out of 35 eyes showed qRT?PCR positivity for virus etiology (57.14%). Cytomegalovirus (CMV) was most commonly identified in nine eyes (45%). The mean DNA copy number was 2,68,339.65 copies/mL (range: 90–3205397). DNA copy number significantly correlated with the extent of clinical involvement (P = 0.013); however, there was no correlation between DNA copy number and presenting visual acuity (P = 0.31), macular involvement (P = 0.675), optic nerve involvement (P = 0.14), and development of retinal detachment (P = 0.73). There was a significant correlation between the number of DNA copies and the timing of sampling (P = 0.0005). Samples taken earlier in the course of the disease had higher viral copies than later ones. Conclusion: qRT?PCR is useful in confirming a viral etiology in over 50% of cases of suspected viral retinitis. It correlates well with the extent of clinical involvement and timing of sampling

Application and Prospect of RNA Profiling Analysis in Forensic Body Fluid Identification / 法医学杂志

In forensic physical evidence identification, the accurate identification of the individual origin and their body fluid composition of the biological samples obtained from the crime scene play a critical role in determining the nature of a crime. In recent years, RNA profiling has become one of the fastest developing methods for body fluids identification. Due to the characteristics of tissue or body fluid specific expression, various types of RNA markers have been proven to be promising candidate markers for body fluids identification in previous studies. This review summarizes the research progress of RNA markers in body fluids identification, including the RNA markers that have been effectively verified in current research and their advantages and disadvantages. Meanwhile, this review prospects the application of RNA markers in forensic medicine.

Research Progress of DNA-Based Technologies for Postmortem Interval Estimation / 法医学杂志

In criminal investigations, postmortem interval (PMI) is important information to be inferred in homicide investigations, as well as the focus and the difficulty in forensic pathology research. Because the DNA content in different tissues is relatively constant and shows changes regularly with the extension of PMI, it has become a research hotspot of PMI estimation. This paper reviews the recent progress of PMI estimation technologies including DNA-based single cell gel electrophoresis, image analysis, flow cytometry, real-time fluorescence quantitative PCR and high-throughput sequencing, hoping to provide references for forensic medicine practice and scientific research.

Reference gene screening for Real-time quantitative PCR in Polygonum multiflorum / 中国中药杂志

To select suitable references gene of Polygonum multiflorum for gene expression analysis in different tissues, five candidate reference genes like Actin,GAPDH,SAND,PP2A,TIP41 were selected from the transcriptome data of P. multiflorum, then the specific primers were designed. The expression stability of the five reference genes in different tissues of P. multiflorum was analyzed by Real-time quantitative PCR through avilable analysis methods such as geNorm, NormFinder, BestKeeper, Delta CT and RefFinder, to ensure the reliability of the analysis results. The results showed that there were significant differences in the expression levels and stability of candidate genes in different tissues of P. multiflorum. Ct distribution analysis of the expression levels of candidate genes showed that the expression levels of Actin and GAPDH genes were relatively high in different tissues, while the expression levels of SAND, PP2A and TIP41 were lower. The stability of each candidate gene was analyzed by different methods. The results of geNorm analysis showed that the expression of PP2A and GAPDH was the most stable, the expression stability of SAND was the worst, the stability of PP2A was the highest in both NormFinder and Delta CT, the stability of SAND was the lowest, and the stability of Actin was the most stable in BestKeeper analysis. Through the comprehensive evaluation and analysis of the stability of candidate genes by RefFinder, it is concluded that the stability of PP2A gene is the highest, followed by GAPDH, Actin, TIP41, SAND, and SAND gene is the worst. Therefore, the PP2A gene is an ideal reference gene for the analysis of gene expression in different tissues of P. multiflorum.

Detection of measles virus infection and proliferation by real-time quantitative PCR / 上海预防医学

Objective A SYBR-Green real-time quantitative PCR(RT-qPCR)method was set up to detect the infection and proliferation of measles virus, which could be useful in virus titer determination. Methods In this project, we used a 405 bp fragment of the N gene of measles virus as a target sequence and constructed a plasmid to establish the standard curve in absolute quantitative experiment. We then used this method to obtain the proliferation curve of measles virus and to detect the virus proliferation at different MOI. Results There was a linear relationship between the virus copy number and the titer of the measles virus reference at the range of 6 to 2 lgCCID50/mL, with a correlation coefficient (r) of 0.991(P < 0.01). Based on the analysis of virus proliferation curve, measles virus mainly proliferated intracellularly within 48 h after its entering the cell. There was no detected increase in viral RNA level in the first 24 h, suggesting the virus was in a silent period in the cell. After 24 h, the virus expanded in large numbers and entered the exponential growth phase. The intracellular viral RNA level reached the plateau phase after its peak at 96 h. The virus secreted to the outside of the cell entered the exponential growth phase starting from 48 h, peaked at 144 h, then followed by plateau phase. Conclusion A SYBR-Green RT-qPCR method is established and used to monitor virus proliferation. Our result is helpful in understanding of the proliferation and secretion of measles virus in cells and provides experimental basis for detection of live attenuated virus titers.

Selection of reference genes for quantitative real-time PCR in Morinda officinalis / 中草药

Objective: To select the appropriate reference genes for calibrating the quantitative real-time PCR detection of gene expression in different tissues and leaves with different treatments of Morinda officinalis. Methods: With different groups and different processing leaves of M. officinalis as materials, 10 internal genes, including GAPDH, CYP, TUA, Actin and so on, were selected as candidate genes according to the M. officinalis transcriptome data. The expression stability of internal reference genes was analyzed by using real-time fluorescence quantification technique combined with software such as geNorm, NormFinder and BestKeeper, so as to select stable reference genes in different tissues and leaves of M. officinalis with different treatments. Finally, appropriate internal reference genes were selected to analyze the relative expression levels of DXS and DXR genes in different tissues and leaves with different treatments. Results: Internal reference genes GAPDH and UBQ were the most stable in different tissues of M. officinalis, the double internal reference combination of GAPDH + UBQ can more accurately analyze the relative expression levels of target genes in different tissues of M. officinalis, while the most stable reference genes in leaves with different treatments were GAPDH and Actin; The selection of the double reference combination of GAPDH + Actin can ensure the reliability of the target gene expression results. In different tissues of M. officinalis, the relative expression of DXS target gene was in sequence of root < stem < leaf, while the relative expression of DXR was stem < root < leaf. The relative expression levels of DXS and DXR genes in leaves with different treatments were increased compared with those untreated leaves (CK). Conclusion: The selected stable internal reference genes lay a foundation for the subsequent study on the expression of related genes of M. officinalis. Using the combination of two stable internal references to homogenize the target genes is conducive to improving the accuracy of the analysis of the expression of target genes.

Optimization of a fluorescent qPCR detection for RNA of SARS-CoV-2 / 生物工程学报

We optimized a fluorescent quantitative polymerase chain reaction (qPCR) assay system for rapid and real time detection of SARS-CoV-2 RNA. The results show that the lowest dilution of RNA samples used for the detection of SARS-CoV-2 RNA could reach 1/10 000 (the initial value is set as 10 ng/μL). Moreover, the cycle threshold (Ct) for samples of clinically diagnosed COVID-19 was lower than 35 or 40. The sensitivity of this method was satisfactory. The results were consistent with those of the COVID-19 detection kit on the market under the same conditions, but the number of cycles required was shortened by about 2. Therefore, the optimized assay developed in this study can be used in screening and early clinical diagnosis. Our work provides a tool to facilitate rapid clinical diagnosis of COVID-19.

Correlation between DNA Methylation of ZNF772 Promoter Region and Cervical Cancer / 中国医学科学院学报

To investigate the DNA methylation in ZNF772 promoter region and its mRNA and protein expressions and analyze the clinical significance of DNA methylation of ZNF772 gene in cervical cancer. Cervical squamous cell carcinoma (SCC) tissues were harvested from three patients (SCC group),and normal cervical tissues from healthy individuals of the same age were used as the control group. Hyper-methylation and lower transcripts were screened by whole-genome bisulfite sequencing (WGBS) and RNA sequencing. Furthermore,in 40 cervical tissue samples in SCC group and 45 normal cervical tissues in the control group,DNA methylation status and mRNA expression of ZNF772 were measured by using real-time quantitative polymerase chain reaction (RT-qPCR) and bisulfite sequencing polymerase chain reaction (BSP). The protein expression was detected by immunohistochemistry. In the SCC group,the potential relationships of DNA methylation status in ZNF772 promoter and mRNA expression with the clinicopathological parameters of cervical cancer were analyzed. As shown by WGBS and RNA sequencing,the abnormal DNA methylated gene ZNF772 was associated with mRNA expression. RT-qPCR verified that the mRNA expression of ZNF772 was significantly lower in SCC group than in control group (=8.351,=0.016). Immunohistochemistry further confirmed that the positive expression of ZNF772 protein was down-regulated in SCC group (=3.802,=0.005). BSP showed that the DNA methylation rate of ZNF772 promoter region (-420,-422 locus) in SCC group was significantly higher than that in control group (=8.566,=0.038;=6.332,=0.043). Spearman correlation analysis showed that,in SCC group,DNA hypermethylation in ZNF772 promoter was negatively correlated with the mRNA expression (=-0.351,=0.045;=-0.349,=0.032) and was significantly correlated with HPV16/18 infection,tumor size,World Health Organization pathological grade,and International Federation of Gynecology and Obstetrics clinical stage (=0.018,=0.012,=0.009,and =0.035,respectively). The DNA hypermethylation in the promoter region of ZNF772 gene is involved in the occurrence and development of cervical cancer.

Selection and validation of internal reference genes for qPCR in Polygonatum cyrtonema tubers at different development stages and in response to abiotic stress / 中国中药杂志

In order to analyze the expression of genes involved in steroidal saponin biosynthesis pathway in Polygonatum cyrtonema tubers, it is very important to select internal reference genes that are stably expressed at different development stages and in response to abiotic stress. According to the previously established P. cyrtonema transcriptome database and reported internal reference genes in plant, this study systematically analyzed eight candidate internal reference genes including histone H2 A, glyceraldehyde-3-phosphate dehydrogenase, ACTIN, β-tubulin, ubiquitin-conjugating enzyme-E2-10, elongation factor 1-alpha isoform, 18 S rRNA and α-tubulin 4 for expression stability in P. cyrtonema tubers at different development stages and in response to methyl jasmonate(MeJA) stress by using Real time fluorescence quantitative PCR(qPCR). Based on the statistical analysis of qPCR results by using GeNorm, NormFinder and BestKeeper softwares, the expression of ubiquitin-conjugating enzyme-E2-10 and elongation factor 1-alpha isoform are the most stable in P. cyrtonema tubes at different development stages and in response to MeJA stress. The two internal reference genes were further validated by analyzing the expression of 4 genes involved in steroidal saponin biosynthesis pathways. In conclusion, ubiquitin-conjugating enzyme-E2-10 and elongation factor 1-alpha isoform can be used as the most appropriate internal reference genes for qPCR analysis in P. cyrtonema. This study also provide a foundation for future investigate the molecular mechanism of steroidal saponin biosynthesis pathways in P. cyrtonema.

Diagnostic ability of real-time quantitative polymerase chain reaction versus immunohistochemistry for Ki-67 assessment in breast cancer: An Indian perspective

Background & objectives: Breast cancer is the most common cancer of women. Inferior prognosis in some patients has been attributed to the higher proliferative capability of the tumour. Immunohistochemistry (IHC) for Ki-67, despite being a simple and cost-effective method, has not become a valid tool to evaluate this biomarker. This is ascribed to variation in pre-analytical and analytical techniques, variable expression, hotspot distribution and inter-and intra-observer inconsistency. This study was aimed at defining the analytical and clinical validity of real-time quantitative polymerase chain reaction (RT-qPCR) as an alternative to IHC evaluation. Methods: This study included a total of 109 patients with invasive breast cancers. Ki-67 IHC visual assessment was compared with the mRNA value determined by RT-qPCR. Concordance between both the methods was assessed. Receiver operating characteristic (ROC) curve analysis and Cohen's kappa value with intraclass correlation were performed. Results: The threshold value for Ki-67 by RT-qPCR obtained by ROC curve was 22.23 per cent, which was used to divide breast cancer cases into high proliferative and low proliferative groups. A significant correlation was observed between both the breast cancer groups formed using RT-qPCR threshold as well as median laboratory value of Ki-67 labelling index by IHC. Interpretation & conclusions: The study results showed a significant correlation between the two methods. While IHC is subject to technical and interpretative variability, RT-qPCR may offer a more objective alternative.

Phaseolus vulgaris genome possesses CAMTA genes, and phavuCAMTA1 contributes to the drought tolerance

Calmodulin-binding transcription activators (CAMTAs) are a family of transcription factors that play an important role in plants’ response to the various biotic and abiotic stresses. The common bean (Phaseolus vulgaris L.) is one of the most important crops in the world and plays a pivotal role in sustainable agriculture. To date, the composition of CAMTA genes in genomes of Phaseolus species and their role in resistance to drought stress are not known. In this study, five PhavuCAMTA genes were characterized in common bean genome through bioinformatics analysis, the morphological and biochemical response of 23 Ph.vulgaris genotypes to different levels of drought stress were evaluated and the expression patterns of PhCAMTA1 in the leaf tissues of sensitive and tolerant genotypes were analysed. Gene structure, protein domain organization and phylogenetic analyses showed that the CAMTAs of Phaseolus were structurally similar and clustered into three groups as other plant CAMTAs. Genotypes showeda differential response to drought stress. Thus, the plant height, number of nodes and flower, total chlorophyll and total protein content, and activity of antioxidant enzymes (ascorbate peroxidase and catalase) in plants significantly influenced by genotype and drought stress interaction. Moreover, the resistant and susceptible genotypes were identified according to three-dimensional plots and the expression patterns of PhavuCAMTA1 gene were studied using real-time quantitative polymerase chain reaction. The results of the present study serve as the basis for future functional studies on the Phaseolus CAMTA family.

Identification of stable reference gene by qRT-PCR in Senecio scandens / 中国中药杂志

As a traditional Chinese medicine, Senecio scandens is rich in important compounds such as flavonoid and sesquiterpenoid. Based on the transcriptome data of S. scandens, 15 candidate reference genes were selected including ABCT, ACT1, ACT2, ACT3, ACBP, ARF, ATPS, EF-H, EF-1α, ETIF, GAPDH, GTPB, MPS, UCE and 60S. Firstly, 9 candidate genes with relatively stable expressions such as ACT1, ACBP, ARF, ATPS, EF-1α, GAPDH, MPS, UCE and 60S were screened from different tissues of S. scandens by RT-PCR. Then, qRT-PCR was used to quantitatively analyze gene expression of these nine candidates in S. scandens with or without stress treatments. Further analysis of these gene expression data by geNorm and NormFinder showed that ACT1 exhibited the stablest expression in all samples and could serve as a reference gene for future study of S. scandens, and provide an endogenous control for gene expression analysis.

An interlaboratory comparison study on the detection of RUNX1-RUNX1T1 fusion transcript levels and WT1 transcript levels / 中华血液学杂志

Objective@#To investigate the current status and real performance of the detection of RUNX1-RUNX1T1 fusion transcript levels and WT1 transcript levels in China through interlaboratory comparison.@*Methods@#Peking University People’s Hospital (PKUPH) prepared the samples for comparison. That is, the fresh RUNX1-RUNX1T1 positive (+) bone morrow nucleated cells were serially diluted with RUNX1-RUNX1T1 negative (-) nucleated cells from different patients. Totally 23 sets with 14 different samples per set were prepared. TRIzol reagent was added in each tube and thoroughly mixed with cells for homogenization. Each laboratory simultaneously tested RUNX1-RUNX1T1 and WT1 transcript levels of one set of samples by real-time quantitative PCR method. All transcript levels were reported as the percentage of RUNX1-RUNX1T1 or WT1 transcript copies/ABL copies. Spearman correlation coefficient between the reported transcript levels of each participated laboratory and those of PKUPH was calculated.@*Results@#①RUNX1-RUNX1T1 comparison: 9 samples were (+) and 5 were (-) , the false negative and positive rates of the 20 participated laboratories were 0 (0/180) and 5% (5/100) , respectively. The reported transcript levels of all 9 positive samples were different among laboratories. The median reported transcript levels of 9 positive samples were from 0.060% to 176.7%, which covered 3.5-log. The ratios of each sample’s highest to the lowest reported transcript levels were from 5.5 to 12.3 (one result which obviously deviated from other laboratories’ results was not included) , 85% (17/20) of the laboratories had correlation coefficient ≥0.98. ②WT1 comparison: The median reported transcript levels of all 14 samples were from 0.17% to 67.6%, which covered 2.6-log. The ratios of each sample’s highest to the lowest reported transcript levels were from 5.3-13.7, 62% (13/21) of the laboratories had correlation coefficient ≥0.98. ③ The relative relationship of the reported RUNX1-RUNX1T1 transcript levels between the participants and PKUPH was not always consistent with that of WT1 transcript levels. Both RUNX1-RUNX1T1 and WT1 transcript levels from 2 and 7 laboratories were individually lower than and higher than those of PKUPH, whereas for the rest 11 laboratories, one transcript level was higher than and the other was lower than that of PKUPH.@*Conclusion@#The reported RUNX1-RUNX1T1 and WT1 transcript levels were different among laboratories for the same sample. Most of the participated laboratories reported highly consistent result with that of PKUPH. The relationship between laboratories of the different transcript levels may not be the same.

Effect of endophytic fungi on expression of key enzyme genes in polysaccharide and alkaloid synthesis from Dendrobium officinale / 中草药

Objective: To analyze the effect of endophytic fungi GXRz2, GXRz3 and GXRz10 on content of poly saccharide and alkaloid, and the expression of key enzyme genes UGPase, HMGR, and FPS in Dendrobium officinale. Methods: The endophytic fungi liquid were added to D. officinale seedlings. Polysaccharide and alkaloid content were measured by spectrophotometry method. With 18S rRNA as internal control gene, the expression of key enzyme genes was detected by real-time quantitative PCR method. Results: It was found that the content of polysaccharide in D. officinale was higher, mainly concentrated in stem, but the lowest in root. And the content of alkaloid in D. officinale was lower, mainly accumulated in leaf, but the lowest in root. In addition, the three endophytic fungi strains could promote accumulation of polysaccharide and alkaloid in D. officinale to a certain extent. The expression of UGPase, HMGR and FPS genes in D. officinale induced by different strains was detected by real-time quantitative PCR. The results showed that endophytic fungi GXRz3 and GXRz10 could significantly increase the expression of UGPase, HMGR and FPS genes in D. officinale. In the polysaccharide synthesis pathway, the UGPase gene had the highest relative expression in the stem, followed by the leaf, and the least in the root. In the alkaloid synthesis pathway, HMGR gene had the highest relative expression in the stem, followed by the leaf, and the least in the root. However, FPS gene had the highest relative expression in the leaf, followed by the stem, and the least in the root. Conclusion: Endophytic fungi may affect the synthesis of polysaccharide by regulating the expression of UGPase. Considering the accumulation of polysaccharide, it is speculated that UGPase may be a key enzyme in the polysaccharide synthesis pathway of D. officinale. Endophytic fungi may affect the synthesis of alkaloid by regulating the expression of HMGR and FPS. Considering the accumulation of alkaloid, it is speculated that FPS may be a key enzyme in the alkaloid synthesis pathway of D. officinale.

Expression and clinical significance of hsa-miRNA-200c in patients with liver metastasis of epithelial ovarian cancer / 中国实用妇科与产科杂志

OBJECTIVE: To investigate the expressions of hsa-miRNA-200 c and its relationship with metastasis of EOC.METHODS: The expression of hsa-miRNA-200 c was detected by Stem-loop Real-time Quantitative PCR(TaqMan probe method)in 73 cases of EOC,30 cases of benign ovarian epithelial tumors and 30 cases of normal ovarian tissues,which were collected in gynecological operations from Guangdong General Hospital from October 2010 to May 2011.Meantime,the clinical pathologic features data were analyzed.The assessment of the correlation between hsa-miRNA-200 c and clinicopathological features,and the hierarchical analysis of hsa-miRNA-200 c level in 73 cases of ovarian epithelial cancer was further undertaken(Among 73 cases,13 patients suffered liver metastasis and 60 patients had non-liver metastasis).Overexpression or knockdown of hsa-miRNA-200 c,ovarian cancer cell invasion and migration abilitywas detected.RESULTS: The expression of miRNA-200 c in the EOC tissues was 382.18±15.22,which was significantly higher than that in the benign ovarian epithelial tumors(35.61 ± 1.42)and normal ovarian tissues(4.43 ±2.23)(P0.05).The expressions of miRNA-200 c were low in EOC with late clin-ical FIGO stage(670.91±16.88 vs. 129.52±33.3,P0.05).The transwell cabinet invasion experiment showed the expression of miRNA-200 c was negatively correlated with the invasion capability of ovarian cancer cells.CONCLUSION: MiRNA-200 c is likely to play a double regulation role in the development of EOC,whose low-expression has been associated with late EOC,lymph node metastasis,liver metastasis,and poor prognosis.

Evaluation of the transcriptional immune biomarkers in peripheral blood from warao indigenous associate with the infection by mycobacterium tuberculosis

Abstract INTRODUCTION: Biomarkers are critical tools for finding new approaches for controlling the spread of tuberculosis (TB), including for predicting the development of TB therapeutics, vaccines, and diagnostic tools. METHODS: Expression of immune biomarkers was analyzed in peripheral blood cells stimulated and non-stimulated with M. tuberculosis antigens ESAT-6, CFP10 and TB7.7. in Warao indigenous individuals. These biomarkers may be able to differentiate TB states, such as active tuberculosis (ATB) cases and latent tuberculosis infection (LTBI) from non-infected controls (NIC). A real-time reverse transcription polymerase chain reaction (RT-qPCR) assay was performed on 100 blood samples under non-stimulation or direct ex vivo conditions (NS=50) and stimulation conditions (S=50). RESULTS: The findings are shown as the median and interquartile range (IQR) of relative gene expression levels of IFN-γ, CD14, MMP9, CCR5, CCL11, CXCL9/MIG, and uPAR/PLAUR immune biomarkers. MMP9 levels were significantly higher in the LTBI-NS and LTBI-S groups compared with the NIC-NS and NIC-S groups. However, CCR5 levels were significantly lower in the LTBI-S group compared with both NIC-NS and NIC-S groups. CCL11 levels were significantly lower in the LTBI-S group compared with the NIC-NS group. CONCLUSIONS: Preliminary findings showed that MMP9 immune biomarkers separated LTBI indigenous individuals from NIC indigenous individuals, while CCR5, CCL11, CD14, and IFN-γ did not differentiate TB states from NIC. MMP9 may be useful as a potential biomarker for LTBI and new infected case detection among Warao indigenous individuals at high risk of developing the disease. It may also be used to halt the epidemic, which will require further validation in larger studies.

Utility Of Real Time Quantitative Pcr In The Diagnosis Of Congenital And Perinatal Cytomegalovirus Infection In Symptomatic Babies

Cytomegalovirus(CMV) infection may be acquired congenitally, perinatally or postnatally in babies. Congenital and perinatal CMV infection can be diagnosed by viral culture or detection and quantitation of CMV DNAby Real Time Quantitative PCR (RT-qPCR) in blood ,urine and body fluids. The objective of this study was to diagnose and determine CMV load in infants presenting with clinical features suggestive of cytomegalovirus infection by RT-qPCR of urine. This descriptive study was done on babies admitted to the Departments of Neonatology and Paediatrics Govt Medical College, Kozhikode from January 2015 to December 2017. Urine samples from 142 babies were received and processed in the Microbiology Department. DNA isolation and amplification was performed using commercial DNA extraction kit and PCR kit for detection and quantification of CMV. Serum samples of the babies with CMV viruria were tested for CMV IgM antibodies. Of 142 babies suggestive of CMVinfection CMV-DNAwas detected and quantitated in urine of 25 (17.60%) (mean age 3.36 months). . CMVIgM was positive in 15/25(60%) babies with viruria .Twenty two had congenital CMV infection (cCMV) and 3 had perinatal infection.The most common clinical presentation was jaundice 13( 52%). Of 8 babies started on Ganciclovir 7 responded to treatment. RT-qPCR helps in diagnosing and quantitating CMVload which helps in deciding on therapy and assessing response to treatment,and can predict risk for long term sequelae.