real time pcr
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Our study aimed to discuss the mechanism of miR-486-3p in controlling the apoptosis of endometrial carcinoma (EC) cells. EC cells were divided into NC group, miR-486-3p mimic and miR-486-3p inhibitor group followed by analysis of miR-486-3p level by Real-time PCR, cell proliferation by spectrophotometric method, apoptosis by FCM, cell migration and invasion by Transwell analysis. EC cells showed reduced miR-486-3p level. The EC malignant biological behaviors could be prompted through retraining miR-486-3p level with increased EC cell invasive capacity. DDR1 was a target of miR-486-3p. The variation of tumor activity could be regulated through controlling DDR1 expression. In conclusion, the apoptotic and invasive characteristic of EC cells are restrained after overexpression of miR-486-3p in EC cells through targeting DDR1, indicating that miR-486-3p could be considered to be one kind of brand-new target for the treatment of EC.

Abstract Background Real-time PCR system is a valuable scientific mainstream needed for quantifying specific gene expression. Nevertheless, compared with conventional PCR, the real-time PCR system is extremely expensive and not affordable for limited or mid-budget research laboratories. Here, a novel, doable and low-cost recipe (referred to as gel express) is developed to quantify gene expression using conventional RT-PCR assay. The novelty of the gel express method is based on replacing crossing point (CP) values with integrated density (IntDen) values of PCR amplicon bands in real-time PCR regular mathematical formulas. Results In this work, gene expression profiles of two different rice stress-marker genes (OsCYP94C2a and OsLOX8) were quantified in response to mechanical wounding at different time points (0, 30, 60, and 150 min). In the gel express method, the free software ImageJ was employed to measure integrated density (IntDen) values of PCR amplicon bands in agarose gel images. IntDen values were then used instead of crossing point (CP) values according to the following modified formula: [EIntDen(ref)/EIntDen(target)]sample ÷ [EIntDen(ref)/EIntDen(target)]control. Gene relative expression profiles (dynamic expression pattern) quantified by gel express method in both genes were highly comparable with real-time RT-PCR. R2 values were 0.9976 and 0.9975 in OsCYP94C2a and OsLOX, respectively. PCR amplification efficiency (E) for all studied genes could be calculated depending on IntDen values through experimentally designed calibration curves. PCR amplification efficiencies with all studied genes obtained by gel express were all in the accepted range. For better-visualized PCR amplicons thus detectable biological effects between treatments, the number of PCR cycles applied in gel express method (IntCyc) was experimentally estimated to be 29 cycles. Conclusions Gel express is a novel, cost-effective and feasible recipe for quantifying gene relative expression in conventional RT-PCR. The expression pattern quantified by gel express is highly comparable and fits the expression data revealed by the used real-time PCR system.

The brown planthopper (BPH), Nilaparvata lugens, is a serious pest of rice throughout Asia. Yeast-like symbionts (YLS) are endosymbionts closely linked with the development of BPH and the adapted mechanism of BPH virulence to resistant plants. In this study, we used semi-quantitative DGGE and absolute quantitative real-time PCR (qPCR) to quantify the number of the three YLS strains (Ascomycetes symbionts, Pichia-like symbionts, and Candida-like symbionts) that typically infect BPH in the nymphal stages and in newly emerged female adults. The quantities of each of the three YLS assessed increased in tandem with the developing nymphal instar stages, peaking at the fourth instar stage, and then declined significantly at the fifth instar stage. However, the amount of YLS present recovered sharply within the emerging adult females. Additionally, we estimated the quantities of YLS for up to eight generations after their inoculation onto resistant cultivars (Mudgo, ASD7, and RH) to reassociate the dynamics of YLS with the fitness of BPH. The minimum number of each YLS was detected in the second generation and gradually increased from the third generation with regard to resistant rice varieties. In addition, the Ascomycetes symbionts of YLS were found to be the most abundant of the three YLS strains tested for all of the development stages of BPH.

Genotyping of single nucleotide polymorphisms (SNPs) is an important task in medicine, veterinary medicine and biology. Precise differentiation of SNPs can be challenging. Methods based onTaqman can lead to false positive results due to nonspecific annealing of the probe. The aim of this research was to develop a new approach for the accurate differentiation of SNPs based on real-time PCR with Taqmanprobes and their rivals.The rivals competed with the Taqmanprobes for annealing to the site. The rivals blocked the nonspecific allele so that the Taqmanprobe could not anneal to it. Thus,the Taqmanprobe only detected specific alleles.This approach madeit possible to fine-tune the diagnostic system by selecting the ratio of Taqmanprobes and rivals (in non-equimolar amounts too).The new approach was tested on several diagonally significant SNPs in veterinary medicine.Using Taqman probes and rival probes showed a significantly greater specificity and efficiency in the determination of both homozygotes and heterozygotes than when conventional systems based only on Taqmanwere used. Keywords: SNP, allele identification, real-time PCR, fluorescent dye

Osteoporosis is a common systemic bone disease caused by the imbalance between osteogenic activity and osteoclastic activity. Aged women are at higher risk of osteoporosis, partly because of estrogen deficiency. However, the underlying mechanism of how estrogen deficiency affects osteoclast activity has not yet been well elucidated. In this study, GSE2208 and GSE56815 datasets were downloaded from GEO database with 25 PreH BMD women and 25 PostL BMD women in total. The RRA algorithm determined 38 downregulated DEGs and 30 upregulated DEGs. Through GO analysis, we found that downregulated DEGs were mainly enriched in myeloid cell differentiation, cytokine-related functions while upregulated DEGs enriched in immune-related biological processes; pathways like Notch signaling and MAPK activation were found in KEGG/Rectome pathway database; a PPI network which contains 66 nodes and 91 edges was constructed and three Modules were obtained by Mcode; Correlation analysis helped us to find highly correlated genes in each module. Moreover, three hub genes FOS, PTPN6, and CTSD were captured by Cytohubba. Finally, the hub genes were further confirmed in blood monocytes of ovariectomy (OVX) rats by real-time PCR assay. In conclusion, the integrative bioinformatics analysis and real-time PCR analysis were utilized to offer fresh light into the role of monocytes in premenopausal osteoporosis and identified FOS, PTPN6, and CTSD as potential biomarkers for postmenopausal osteoporosis.