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Quantitative RT-PCR

Wilson Huanca-Mamani, Raúl Arias-Carrasco, Steffany Cárdenas-Ninasivincha, Marcelo Rojas-Herrera, Gonzalo Sepúlveda-Hermosilla, José Carlos Caris-Maldonado, Elizabeth Bastías, Vinicius Maracaja-Coutinho
Long non-coding RNAs (lncRNAs) have been defined as transcripts longer than 200 nucleotides, which lack significant protein coding potential and possess critical roles in diverse cellular processes. Long non-coding RNAs have recently been functionally characterized in plant stress-response mechanisms. In the present study, we perform a comprehensive identification of lncRNAs in response to combined stress induced by salinity and excess of boron in the Lluteño maize, a tolerant maize landrace from Atacama Desert, Chile...
March 20, 2018: Genes
Masoumeh Nemati, Naser Ajami, Mehrdad Asghari Estiar, Saleheh Rezapour, Reyhaneh Ravanbakhsh Gavgani, Shahryar Hashemzadeh, Hossein Samadi Kafil, Ebrahim Sakhinia
BACKGROUND: To date, 4 classes of histone deacetylases (HDACs) have been identified in humans. Class I HDACs are zinc-dependent and NAD+-independent enzymes, and include 4 isoforms closely related to yeast RPD3: HDAC1, 2, 3, and 8. OBJECTIVES: The aims of the study were to quantitatively evaluate the expression of HDAC3 in colorectal cancer (CRC) and to correlate its expression levels with clinicopathological parameters. MATERIAL AND METHODS: We characterized expression patterns of HDAC3 as class I HDAC isoforms in a cohort of 48 CRC patients by quantitative (real-time) reverse transcription polymerase chain reaction (RT-PCR)...
March 20, 2018: Advances in Clinical and Experimental Medicine: Official Organ Wroclaw Medical University
Bo Wang, Shuangshuang Han
Traumatic brain injury (TBI), resulting from external force on the head, usually leads to long-term deficits in motor and cognitive functions. Inducible nitric oxide synthase (iNOS)-mediated excessive inflammation could exacerbate brain damage after TBI. The present study therefore investigated the potential neuroprotective effects of iNOS inhibition after TBI. Male C57BL/6J mice were subjected to controlled cortical impact injury and then treated with high selective iNOS inhibitor 1400W. Expression of iNOS mRNA was determined by quantitative RT-PCR...
March 20, 2018: Cerebellum
Liping Huang, Surapun Tepaamorndech, Catherine P Kirschke, John W Newman, William R Keyes, Theresa L Pedersen, Jureeporn Dumnil
ZnT7 (Slc30a7) is a widely expressed zinc transporter involved in sequestration of zinc into the Golgi apparatus and vesicular compartments. Znt7-knockout (KO) mice are mildly zinc deficient and lean. Despite their lean phenotype, adult male Znt7-KO mice are prone to insulin resistance. We hypothesized that fat partitioning from adipose to non-adipose tissues causes insulin resistance in Znt7-KO mice. Here, we used biological and biochemical methods including fatty acid and oxylipin profiling, electron microscopy, immunohistochemistry, quantitative RT-PCR, and Western blot analysis to identify the underlying mechanism of insulin resistance in Znt7-KO mice...
March 19, 2018: Journal of Biological Chemistry
Waqas Ahmed, Maria Razzaq
Brucella spp. are facultative intracellular pathogens responsible for brucellosis, an emerging zoonosis that results in abortion, sterility in host animals, and chronic relapsing fever in humans. The small noncoding RNA (sRNA) AbcR is a textbook example of a molecule acting on trans-encoded target mRNAs, a principal element of bacterial gene regulation required for wild-type virulence in α-proteobacteria. Here, we used bioinformatics and experimental validation to characterize the interaction between AbcR sRNA and target mRNA...
March 16, 2018: Microbial Pathogenesis
P Scott Pine, Steven P Lund, Sanford A Stass, Debra Kukuruga, Feng Jiang, Lynn Sorbara, Sudhir Srivastava, Marc Salit
BACKGROUND: We demonstrate the feasibility of creating a pair of reference samples to be used as surrogates for clinical samples measured in either a research or clinical laboratory setting. The reference sample paradigm presented and evaluated here is designed to assess the capability of a measurement process to detect true differences between two biological samples. Cell-based reference samples can be created with a biomarker signature pattern designed in silico. Clinical laboratories working in regulated applications are required to participate in proficiency testing programs; research laboratories doing discovery typically do not...
March 20, 2018: BMC Biotechnology
Stefanie Gerson, Jennifer Nowak, Esther Zander, Julia Ertel, Yurong Wen, Oleg Krut, Harald Seifert, Paul G Higgins
Objectives: To investigate the mechanisms of tigecycline resistance in isogenic Acinetobacter baumannii isolate pairs as well as 65 unique clinical A. baumannii isolates obtained during the MagicBullet clinical trial from Greece, Italy and Spain. Methods: A. baumannii isolates were subjected to WGS and the regulatory genes of resistance-nodulation-cell division (RND)-type efflux pumps were analysed. MICs were determined by agar dilution and the expression of RND-type efflux pumps was measured by semi-quantitative RT-PCR...
March 14, 2018: Journal of Antimicrobial Chemotherapy
Takuma Iguchi, Noriyo Niino, Satoshi Tamai, Ken Sakurai, Kazuhiko Mori
RT-qPCR is one of the most common methods to assess individual target miRNAs. MiRNAs levels are generally measured relative to a reference sample. This approach is appropriate for examining physiological changes in target gene expression levels. However, absolute quantification using better statistical analysis is preferable for a comprehensive assessment of gene expression levels. Absolute quantification is still not in common use. This report describes a protocol for measuring the absolute levels of plasma miRNA, using RT-qPCR with or without pre-amplification...
February 12, 2018: Journal of Visualized Experiments: JoVE
Fangfang Tao, Xinxin Tian, Zhiqian Zhang
Long non-coding RNAs (lncRNAs) have been reported to be of great importance in the formation and progression of a wide range of human carcinomas including prostate cancer (PCa). Among them, PCAT3 and PCAT9 have been identified as two prostate tissue-specific lncRNAs and are up-regulated in PCa. However, their roles in the biological characteristics of PCa have not been fully elucidated. In the present study, our data revealed that knockdown of PCAT3 and PCAT9 suppressed cellular proliferation, invasion, migration, angiogenesis and stemness in androgen-dependent LNCaP and 22Rv1 cells...
February 23, 2018: Oncotarget
Hua Zhang, Yumei Cao, Yuru Chen, Guangxi Li, Hanshu Yu
The present study investigated the inhibitory effects of apatinib on the proliferation of the SMMC-7721 hepatocellular carcinoma cell line to explore the possible mechanism. The MTT assay was used to detect the inhibitory effects of the different concentrations of apatinib on the proliferation of SMMC-7721 cells. Annexin V/PI double staining was performed to investigate the effects of apatinib on the apoptosis of SMMC-7721 cells. Expression of the apoptosis-related genes Bcl-2, Bax and caspase-9 after apatinib treatment was detected by reverse transcription-quantitative PCR (RT-qPCR) and western blot analysis...
April 2018: Oncology Letters
Dong Leng, Ran Miao, Xiaoxi Huang, Ying Wang
Octamer-binding protein 2 (Oct2) binds to the ATGCAAAT octamer on the IgH enhancer and stimulates IgH expression in human multiple myeloma (MM). Cysteine-rich secreted protein 3 (CRISP3) possesses the ATGCAAAT sequence and thus is activated by Oct2 in mouse B cells, suggesting that CRISP3 may be activated in and be a potential biomarker for MM. The present study involved a meta-analysis of the gene expression profiling data of human MM peripheral blood. Significantly expressed genes were analyzed on merged super array microarray data and selected sample data with significantly expressed genes were additionally analyzed by principal component analysis and Bayesian probit regression...
April 2018: Oncology Letters
Chunyan Mo, Shumin Wan, Youquan Xia, Ning Ren, Yang Zhou, Xingyu Jiang
Cassava is an energy crop that is tolerant of multiple abiotic stresses. It has been reported that the interaction between Calcineurin B-like (CBL) protein and CBL-interacting protein kinase (CIPK) is implicated in plant development and responses to various stresses. However, little is known about their functions in cassava. Herein, 8 CBL ( MeCBL ) and 26 CIPK ( MeCIPK ) genes were isolated from cassava by genome searching and cloning of cDNA sequences of Arabidopsis CBL s and CIPK s. Reverse-transcriptase polymerase chain reaction (RT-PCR) analysis showed that the expression levels of MeCBL and MeCIPK genes were different in different tissues throughout the life cycle...
2018: Frontiers in Plant Science
Chao Shi, Bei-Qing Pan, Feng Shi, Zhi-Hui Xie, Yan-Yi Jiang, Li Shang, Yu Zhang, Xin Xu, Yan Cai, Jia-Jie Hao, Ming-Rong Wang
Esophageal squamous cell carcinoma (ESCC) is one of the malignancies in digestive system, with a low 5-year survival rate. We previously revealed that Sequestosome 1 (SQSTM1/p62) protein levels were upregulated in ESCC tissues. However, it is unclear about the function of p62 and the underlying mechanism. Here, we used immunofluorescence and immunohistochemistry to investigate the expression of p62 in ESCC. Western blotting, quantitative RT-PCR, colony formation assay, flow cytometry, immunoprecipitation and xenograft tumor assay were used to analyze the role of p62 in vitro and vivo...
March 19, 2018: Oncogene
Ziyao Liu, Yun Tang, Rongrong Zhou, Xiaosa Shi, Hongmei Zhang, Tengfei Liu, Zenglin Lian, Xinyuan Shi
BACKGROUND: The bi-directional solid fermentation product extract of Trametes robiniophila Murr (Huaier) with Radix Isatidis (TIF) has been shown to have good anti-tumor activity. However, the mechanisms of this activity are still unknown. In the present study, we aimed to investigate its inhibitory effect on both SK-BR-3 and MDA-MB-231 cells, and explore the possible mechanisms of its anti-cancer effect in vitro. METHODS: The experiment comprised a control group, Radix Isatidis group, Huaier group, and TIF group...
March 15, 2018: Journal of the Chinese Medical Association: JCMA
Christelle Sanchez, Gabriel Mazzucchelli, Cécile Lambert, Fanny Comblain, Edwin DePauw, Yves Henrotin
OBJECTIVE: Osteoarthritis (OA) is characterized by cartilage degradation but also by other joint tissues modifications like subchondral bone sclerosis. In this study, we used a proteomic approach to compare secretome of osteoblast isolated from sclerotic (SC) or non sclerotic (NSC) area of OA subchondral bone. DESIGN: Secretome was analyzed using differential quantitative and relative label free analysis on nanoUPLC G2 HDMS system. mRNA of the more differentially secreted proteins were quantified by RT-PCR in cell culture from 5 other patients...
2018: PloS One
Naoya Ishibashi, Tatsuaki Watanabe, Masahiko Kanehira, Yui Watanabe, Yasushi Hoshikawa, Hirotsugu Notsuda, Masafumi Noda, Akira Sakurada, Shinya Ohkouchi, Takashi Kondo, Yoshinori Okada
PURPOSE: Using a rat model of allograft lung transplantation, we investigated the effectiveness of mesenchymal stromal cells (MSCs) as prophylactic and therapeutic agents against the acute rejection of lung grafts. METHODS: Lung grafts were harvested from donor rats and transplanted orthotopically into major histocompatibility complex-mismatched rats. MSCs were administered to the recipients once (on day 0) or twice (on days 0 and 3) after transplantation. The grade of acute rejection was evaluated both macroscopically and microscopically 6 days after transplantation...
March 15, 2018: Surgery Today
Natalia Anahí Juiz, Irma Torrejón, Marianela Burgos, Ana María Fernanda Torres, Tomás Duffy, Nelly Melina Cayo, Anahí Tabasco, Miriam Salvo, Silvia Andrea Longhi, Alejandro Gabriel Schijman
Infection with Trypanosoma cruzi in women at reproductive age is associated with congenital transmission and adverse pregnancy outcome. The placenta is a key barrier to infection. We characterized gene expression profiles of term placental environment from T. cruzi seropositive (SP) and seronegative (SN) mothers performing RNA-seq. Nine pools of placental RNA paired samples were used: three from SN and six from SP tissues. Each pool consisted of female/male newborns and vaginal/caesarean deliveries binomials...
March 12, 2018: American Journal of Pathology
Quan-Bo Zhang, Yu-Feng Qing, Cong-Cong Yin, Li Zhou, Xian-Shuang Liu, Qing-Sheng Mi, Jing-Guo Zhou
BACKGROUND: MicroRNAs (miRNAs) serve as important regulators of inflammatory and immune responses and are implicated in several immune disorders including gouty arthritis. The expression of miR-146a is upregulated in the peripheral blood mononuclear cells of patients with inter-critical gout when compared to normouricemic and hyperuricemic controls and those patients with acute gout flares. However, the role of miR-146a in the development of gout remains unknown. Here, we used miR-146a knockout (KO) mice to test miR-146a function in a monosodium urate (MSU)-induced gouty arthritis model...
March 15, 2018: Arthritis Research & Therapy
Laura Sangl, Kaspar Matiasek, Sandra Felten, Stefanie Gründl, Michele Bergmann, Hans-Jörg Balzer, Nikola Pantchev, Christian M Leutenegger, Katrin Hartmann
Objectives The amino acid substitutions M1058L and S1060A in the spike protein of feline coronavirus (FCoV) have been postulated to be responsible for the development of the pathogenic feline infectious peritonitis virus (FIPV), which causes feline infectious peritonitis (FIP). The aim of the following study was to investigate the presence of mutated virus in tissue samples of cats with and without FIP. Methods The study population consisted of 64 cats, 34 of which were diagnosed with FIP and 30 control cats...
March 1, 2018: Journal of Feline Medicine and Surgery
Cosimo Cumbo, Luciana Impera, Crescenzio Francesco Minervini, Paola Orsini, Luisa Anelli, Antonella Zagaria, Nicoletta Coccaro, Giuseppina Tota, Angela Minervini, Paola Casieri, Claudia Brunetti, Antonella Russo Rossi, Elisa Parciante, Giorgina Specchia, Francesco Albano
For monitoring minimal residual disease (MRD) in chronic myeloid leukemia (CML) the most recommended method is quantitative RT-PCR (RT-qPCR) for measuring BCR-ABL1 transcripts. Several studies reported that a DNA-based assay enhances the sensitivity of detection of the BCR-ABL1 genomic rearrangement, even if its characterization results difficult. We developed a DNA-based method for detecting and quantifying residual BCR-ABL1 positive leukemic stem cells in CML patients. We propose two alternative approaches: the first one is a fluorescence in situ hybridization (FISH)-based step followed by Sanger sequencing; the second one employs MinION, a single molecule sequencer based on nanopore technology...
February 16, 2018: Oncotarget
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