Analytical Methods
·
2024
·Vol. 16
·期 8
·1281-1287
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IF 2.7
Keyu Li,Peng Xiao,Ningning Yuan,Shujuan Yan,Pei Zhao,Guowei Zuo
MiRNAs are critical regulators of target gene expression in many biological processes and are considered promising biomarkers for diseases. In this study, we developed a simple, specific, and sensitive miRNA detection method based on proximity ligation reaction, which is easy to operate. The method uses a pair of target-specific DNA probes immobilized on the same gold nanoparticles (AuNPs), which hybridize to the target miRNA. Hybridization brings the probes close together, allowing the formation of a continuous DNA sequence that can be amplified by Quantitative Real-time PCR (qPCR). This method eliminates the need for complex reverse transcription design and achieves high specificity for discriminating single base mismatches between miRNAs through a simple procedure. This method can sensitively measure three different miRNAs with a detection limit of 20 aM, providing high versatility and sensitivity, even distinguishing single-base variations among members of the miR-200 family with high selectivity. Due to its high selectivity and sensitivity, this method has important implications for the investigation of miRNA biological functions and related biomedical research.
研究领域:
分子生物学
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纳米生物技术
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医学检测
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产品关联
DOI
PubMed
高影响因子
Forensic Sciences Research
·
2019
·Vol. 7
·期 2
·172-180
·
IF 0
Tao Ruiyang,Chen Chong,Sheng Xiang,Xia Ruocheng,Zhang Xiaochun,Zhang Jingyi,Yang Zihao,Zhang Suhua,Li Chengtao
The Investigator 24plex QS Kit (QIAGEN, Hilden, Germany) is a 6-dye fluorescent chemistry short tandem repeat (STR) polymerase chain reaction (PCR) amplification system that simultaneously amplifies 20 of the expanded Combined DNA Index System (CODIS) core STR loci, SE33, DYS391, and the standard sex-determining locus, amelogenin, as well as two special internal performance quality sensor controls (QS1 and QS2), which are included in the primer mix to check the PCR performance. This study was designed to be a pilot evaluation of this STR-PCR kit in a Chinese Han population regarding the PCR conditions, sensitivity, precision, accuracy, repeatability, reproducibility, and concordance; tolerance to PCR inhibitors; applicability to real “forensic-type” samples; species specificity; mixture, balance and stutter analyses, and utility in a population investigation. The exhaustive validation studies demonstrated that the Investigator 24plex QS system is accurate, sensitive and robust for STR genotyping. In addition, these genetic markers in the population data in our study indicated that they can also be useful for forensic identification and paternity testing in the Chinese Han population.
研究领域:
法医学
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分子生物学
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遗传学
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产品关联
DOI
PubMed
CELLULAR AND MOLECULAR BIOLOGY
·
2023
·Vol. 69
·期 9
·
IF 1.6
Aili Xuan,Mei Yang,Qun Xia,Qian Sun
Asthma is a respiratory inflammatory disease, and nicotinamide adenine dinucleotide phosphate oxidase 4 (NOX4) is involved in the progression of respiratory diseases. However, the role of NOX4 in asthma remains unclear. In the present study, we aimed to explore the effects of NOX4 on airway remodeling and inflammation. NOX4 expression was measured using immunocytochemistry (IHC), western blot, and real-time PCR (qPCR). Lung tissues were stained using the H&E assay. ELISA was used to examine the levels of airway remodeling-related indicators, and qPCR was used to detect airway inflammatory factors. The results indicated that NOX4 is highly expressed in lung tissues, bronchoalveolar lavage fluid (BALF), and serum of OVA-treated mice. Inhibition of NOX4 alleviated OVA-induced airway remodeling and inflammation. Similarly, TGF-β1 was also upregulated in BALF and serum OVA-induced mice. Inhibition of TGF-β1 signaling also improved airway remodeling and inflammation induced by OVA. Moreover, the downregulation of NOX4 inactivated the TGF-β1-Smad2/3 pathway, and TGF-β1 decreased Smad2/3 expression. Moreover, inhibition of the TGF-β1 was enhanced, while TGF-β1 reversed the effects on airway remodeling and inflammation induced by NOX4 inhibition. Taken together, the downregulation of NOX4 improves airway remodeling and inflammation via inactivation of the TGF-β1-Smad2/3 pathway in asthma mice, suggesting that NOX4 may be a therapeutic target for asthma.
研究领域:
呼吸系统疾病
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分子生物学
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免疫学
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DOI
PubMed
高影响因子
INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES
·
2012
·Vol. 13
·期 12
·17006-17018
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IF 2.598
Beihua Bao,Ting Geng,Yudan Cao,Weifeng Yao,Li Zhang,Anwei Ding
The aim of this study was to find out whether Schizonepetin influences the pharmacokinetics of the main substrates drugs of CYP1A2, CYP3A1/2, CYP2E1, CYP2C19 and CYP2D6 in rats; the influence on the levels of CYP mRNA was also studied. Phenacetin, dapsone, chlorzoxazone, omeprazole and metoprolol were selected as probe substrates for CYP1A2, CYP3A1/2, CYP2E1, CYP2C19 and CYP2D6 respectively. HPLC methods were employed for the determination of these substrates in plasma and the pharmacokinetic parameters were calculated. Real-time RT-PCR was used to determine the effects of Schizonepetin on the mRNA expression of CYP3A1, CYP1A2 and CYP2E1 in the rat liver. After the rats were orally administrated with Schizonepetin once a day for seven consecutive days, there were significant differences in plasma concentration of phenacetin, dapsone, chlorzoxazone and metoprolol, but not omeprazole, as compared with pre-administration. In addition, Schizonepetin induced the expression of CYP3A1, CYP1A and CYP2E1 at dosages of 24 and 48 mg/kg. Our results indicated that Schizonepetin had significant induction effects on CYP3A1/2 and inhibition effects on CYP1A2, CYP2E1 or CYP2D6 as oriented from the pharmacokinetic profiles of the substrates. Moreover, in the mRNA expression levels, Schizonepetin could induce the mRNA expression of CYP3A1, CYP1A and CYP2E1. In conclusion, co-administration of some CYP substrates with Schizonepetin may lead to an undesirable herb-drug interaction.Keywords:Schizonepetin;Cytochrome P450;substrates drugs;pharmacokinetic parameters;mRNA;real-time RT-PCR
研究领域:
药理学
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分子生物学
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DOI
PubMed
高影响因子
ACS Omega
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2022
·
IF 4.132
Xianjing Zhang,Jun Guo,Bo Song,Feng Zhang
The polymerase chain reaction (PCR) has been widely used in medical diagnosis and forensic identification due to its ultrahigh sensitivity and signal amplification. Metal ions (i.e., Cu2+, Zn2+) have been considered PCR inhibitors and rarely shown their positive roles in PCR amplification until our report, in which we discovered that metal ions can significantly improve the PCR specificity and the yield of target DNA sequences. For an in-depth investigation with taking copper ions as a typical model, here we found an interesting spatiotemporal regulation mechanism of metal ions in PCR. The ionic concentration window for improving PCR specificity not only was independent of annealing temperature but also can be well regulated by both the annealing time and extension time. Using the ionic concentration window as a measure, the time affects either the amount or the sequence length of nonspecific amplicons in the space. The mechanism proposed in this work will deepen our understanding of the unneglectable roles of metal ions in DNA replication and meanwhile provide a new strategy for designing regulation kits for PCR-based biomedical applications.
研究领域:
分子生物学
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生物化学
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DOI
PubMed
高影响因子
ACS Applied Materials & Interfaces
·
2023
·
IF 9.5
Zhongjie Fei,Ping Liu,Chu Cheng,Rongbin Wei,Pengfeng Xiao,Youfa Zhang
Although numerous approaches were proposed for the nucleic acid (NA)-based SARS-CoV-2 detection, the nonideal NA desorption efficiency of conventional magnetic beads (MBs) limits their widespread application. In this study, we developed solvent-responsive MBs (called responsive MBs), which, in the presence of buffers, modulated the absorption and desorption capacities of NA by flipping the surface −COO–. Relative to other commercial MBs, responsive MBs exhibited similar absorption profiles and markedly enhanced desorption profiles. When applied for NA detection of complex samples, responsive MBs exhibited better performance of RNA detection than DNA, with obvious advantages in sensitivity. Specifically, the RNA and DNA desorption rates of commercial MBs were ∼85 and 82.5%, while those of responsive MBs were nearly 94 and 93.5%, respectively. Furthermore, responsive MBs exhibited remarkable extraction ability in a wide range of tissues and better performance of RNA extraction than DNA. When applied for SARS-CoV-2 detection, the responsive MBs along with the simulated digital RT-LAMP (a previously established apparatus) further improved detection efficiency, yielding a precise quantitative detection as low as 25 copies and an ultimate sensibility detection of 5 copies/mL. It was also successfully employed in numerous NA-based technologies such as polymerase chain reaction (PCR), sequencing, and so on.
研究领域:
生物技术
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分子生物学
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病毒学
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分析化学
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产品关联
DOI
PubMed
高影响因子
INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES
·
2024
·Vol. 25
·期 17
·9456
·
IF 4.9
Xinru Hou,Kewen Zhang,Yingmin Lyu
Oriental hybrid lilies, known for their vibrant colors, diverse flower shapes, and long blooming seasons, require annual bulb propagation in horticultural production. This necessity can lead to higher production costs and limit their use in landscaping. The LA hybrid lily ‘Aladdin’ has shown strong self-reproduction capabilities in optimal cultivation environments, producing numerous high-quality underground stem bulblets. This makes it a valuable model for studying bulblet formation in lilies under natural conditions. Through transcriptome data analysis of different developmental stages of ‘Aladdin’ bulblets, theLaLBD37gene, linked to bulblet formation, was identified. Bioinformatics analysis, subcellular localization studies, and transcriptional activation activity tests were conducted to understand the characteristics ofLaLBD37. By introducing theLaLBD37gene into ‘Sorbonne’ aseptic seedlings via Agrobacterium-mediated transformation, resistant plants were obtained. Positive plants were identified through various methods such as GUS activity detection, PCR, and fluorescence quantitative PCR. Phenotypic changes in positive plants were observed, and various physiological indicators were measured to confirm the role ofLaLBD37in bulblet formation, including soluble sugar content, starch content, sucrose synthase activity, and endogenous hormone levels. The findings suggest that theLaLBD37gene plays a significant role in promoting the development of lily bulblets, offering insights for enhancing the reproductive capacity of Oriental hybrid lilies and exploring the molecular mechanisms involved in lily bulb regeneration.
研究领域:
植物生物学
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分子遗传学
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园艺学
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DOI
高影响因子
STAR Protocols
·
2023
·
IF 0
Jiaqi Shi,Caiqi Liu,Shengnan Luo,Yingying Zhang,Mingwei Li,Meng Zhou,Mingjiao Weng,Xiaobo Li,Tongsen Zheng
Summary Here, we present a protocol for the detection of the two STING isoforms (erSTING and pmSTING) in human peripheral blood mononuclear cells or mouse splenocytes using Western blot and PCR. We detail steps to construct plasmids encoding each isoform and transfer them into mouse and human cell lines. Finally, we describe how to detect cell membrane localization of pmSTING using flow cytometry, immunoprecipitation, and immunofluorescence. This protocol is applicable for proteins with well-predicted topological structures. For complete details on the use and execution of this protocol, please refer to Li et al. 1 .
研究领域:
分子生物学
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细胞生物学
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免疫学
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DOI
PubMed
JOURNAL OF PHARMACEUTICAL AND BIOMEDICAL ANALYSIS
·
2014
·
IF 2.829
Yajing Lei,Xiaoyi Li,Muhammad Sajid Hamid Akash,Lifang Zhou,Xiaojing Tang,Weixing Shi,Zhiming Liu,Shuqing Chen
Salbutamol (SAL) is known as quick-acting β-2 receptor agonist and its use in humans for pulmonary diseases and/or in animal feed is limited because of associated potential hazardous effects on health. Several techniques are available for the detection of SAL, but are expensive and time consuming. Here for the first time, a novel pre-assembled DNA immunoprobe-based immuno-PCR assay was developed to investigate the levels of SAL in human urine samples and compared the proposed immuno-PCR assay for the detection of SAL with that of direct competitive ELISA. Under the optimized conditions, the assay showed a linear range over seven orders of magnitude, whereas the limit of detection of SAL in buffer was found to be 21 fg/mL. Current immuno-PCR assay exhibited a 300-fold better detection limit for SAL as compared to one achieved with direct competitive ELISA using the same antibody. In conclusion, it has been found that the developed method is highly sensitive and simple method that can significantly enhance the detection sensitivity for small molecules. Moreover it can be modified and used for detecting other small molecules and/or chemicals that exist in the environment and are responsible for the environmental pollution.
研究领域:
分析化学
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分子生物学
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环境科学
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产品关联
DOI
PubMed
高影响因子
Cancer Reports
·
2022
·Vol. 6
·期 2
·e1688
·
IF 0
Ying Liang,Yan Liu,Pin Zhang,Mengxin Zhang,Bang Du,Weyland Cheng,Zhidan Yu,Lifeng Li,Huanmin Wang,Guangjun Hou,Xianwei Zhang,Wancun Zhang
The postoperative recurrence of neuroblastoma (NB) patients is an essential reason for the high mortality of NB due to the lack of early, non-invasive, and dynamic strategies for monitoring NB recurrence. Therefore, whether the plasma circulating cell-free MYCN gene as an indicator for monitoring of NB recurrence was systematically evaluated. The MYCN copy number and NAGK (reference gene) copy number (M/N) ratio in plasma and corresponding tumor tissues of NB patients was detected using an economical, sensitive, and specific single-tube dual RT-PCR approach developed in this study. The plasma M/N ratio of the MYCN gene amplification (MNA) group ( N = 25, median M/N ratio = 4.90) was significantly higher than that of the non-MNA group ( N = 71, median M/N ratio = 1.22), p < .001. The M/N ratio in NB plasma ( N = 60) was positively correlated with the M/N ratio in NB tumor tissue ( N = 60), with a correlation coefficient of 0.9496. In particular, the results of dynamic monitoring of postoperative plasma M/N ratio of NB patients showed that an abnormal increase in M/N ratio could be detected 1–2 months before recurrence in NB patients. In summary, the single-tube double RT-PCR approach can be used to quantitatively detect MYCN copy number. The copy number of MYCN in the tissue and plasma of NB patients is consistent with each other. More importantly, the circulating cell-free MYCN gene of NB patients can be used as a monitoring indicator for early, non-invasive, and dynamic monitoring of NB recurrence.
研究领域:
肿瘤学
·
分子生物学
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遗传学
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DOI
PubMed
ANALYTICA CHIMICA ACTA
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2020
·
IF 5.977
Lei Peng,Jin Zhou,Lijuan Yin,Shuli Man,Long Ma
For the first time, three 2-input elementary AND, OR, INHIBIT logic gates have been constructed by using CRISPR-Cas12a system. These logic gates utilised the intrinsic advantages of programmability, sequence specificity and high base resolution of CRISPR-Cas12a system. Among them, the AND gate owned the potentials as a built-in biosensor that responded rapidly to external pathogenic bacteria such as Staphylococcus aureus with high sensitivity and specificity. We applied the CRISPR-Cas12a based bacterial detection after a target-amplification using PCR. The total sample-to-answer time was appropriately 2.0 h, the limit of detection (LOD) was 10 3 CFU/mL, and the dynamic range was 10 3 –10 7 CFU/mL. Also, the sequence addressability enabled this AND logic gate to accurately trace back and distinguish input genes. These above-mentioned features were highly ideal to incur a rapid response to pathogenic bacteria for decision making. Our results not only validated the possibility of using CRISPR-Cas systems for constructing bio-computing devices but also provided a prototype of biosensor for rapid and intelligent pathogenic bacteria detection.
研究领域:
生物技术
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分子生物学
·
微生物学
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DOI
PubMed
高影响因子
ANALYTICAL LETTERS
·
2023
·
IF 2
Peipei Cao,Xiangde Lai,Rui Zhang,Zhiling Chen,Bin Qiao,Lina Niu,Qiang Wu
Monkeypox, a rare zoonotic disease classified as a category B infectious disease, is caused by the monkeypox virus (MPXV), which has a broad host range. Since May 2022, human MPXV infection outbreak has spread to more than 100 countries, and China has reported 106 MPXV infections between June 2 and 30, 2023. Therefore, rapid and effective MPXV detection is essential to control and prevent its transmission. In this study, a rapid, cost-effective, and convenient fluorescent immunochromatographic assay (FICA) for MPXV antigen A35R detection was developed. Under the optimal reaction conditions, the visual detection limit of the proposed assay was 7.5 ng/mL and was completed within 15 min. There was no cross-reactivity with norovirus (NV) or dengue virus (DENV) controls. Additionally, the clinical application of the proposed method was demonstrated by testing human serum samples spiked with the MPXV antigen A35R. FICA was faster and more convenient than traditional enzyme-linked immunosorbent assays (ELISA) or polymerase chain reaction (PCR), and hence suitable for the early screening of MPXV infection. In summary, FICA could realize rapid, low-cost, sensitive, and specific MPXV detection hold potential application prospects in clinical settings.
研究领域:
病毒学
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传染病
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分子诊断
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临床微生物学
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DOI
高影响因子
危险品化学品经营许可证（带存储）