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https://www.readbyqxmd.com/read/29614692/determination-of-qpcr-reference-genes-suitable-for-normalizing-gene-expression-in-a-canine-model-of-duchenne-muscular-dystrophy
#1
John C W Hildyard, Frances Taylor-Brown, Claire Massey, Dominic J Wells, Richard J Piercy
BACKGROUND: Dogs with dystrophin-deficient muscular dystrophy are valuable models of the equivalent human disease, Duchenne Muscular Dystrophy (DMD): unlike the mdx mouse, these animals present a disease severity and progression that closely matches that found in human patients. Canine models are however less thoroughly characterised than the established mdx mouse in many aspects, including gene expression. Analysis of expression in muscle plays a key role in the study of DMD, allowing monitoring and assessment of disease progression, evaluation of novel biomarkers and gauging of therapeutic intervention efficacy...
March 26, 2018: Journal of Neuromuscular Diseases
https://www.readbyqxmd.com/read/29577185/variability-of-the-expression-of-reference-genes-in-samples-of-human-olfactory-epithelium
#2
S P Kovalenko, N S Rudenko, A S Avdeeva, K V Danilenko, L I Aftanas
We analyzed variability of the expression of three reference genes in biopsy samples of the olfactory epithelium obtained from healthy volunteers. The expression of B2M, HPRT1, and CASC3 genes was analyzed by real-time PCR. The pairs of genes B2M-HPRT1 and B2M-CASC3 were found to possess minimum individual variability of expression and can be reliable candidates for the reference genes in analysis of gene expression in neural cells.
March 26, 2018: Bulletin of Experimental Biology and Medicine
https://www.readbyqxmd.com/read/29512759/selection-of-reference-genes-for-expression-analysis-in-mouse-models-of-acute-alcoholic-liver-injury
#3
Sheng Wang, Jianqing Wang, Xiongwen Lv
Investigations of hepatic gene expression are crucial for determining the molecular factors involved in acute alcoholic liver injury. The results of liver molecular investigations may reveal etiologically important genomic alterations. Therefore, it is necessary to normalize gene expression data to identify stable genes, which may be used as a reference under different experimental conditions. The aim of the present study was to apply reverse transcription‑quantitative polymerase chain reaction analysis and use analysis software to investigate the expression stability of candidate reference genes in hepatic tissues from mice with acute alcoholic liver injury...
March 1, 2018: International Journal of Molecular Medicine
https://www.readbyqxmd.com/read/29507284/microhomology-assisted-scarless-genome-editing-in-human-ipscs
#4
Shin-Il Kim, Tomoko Matsumoto, Harunobu Kagawa, Michiko Nakamura, Ryoko Hirohata, Ayano Ueno, Maki Ohishi, Tetsushi Sakuma, Tomoyoshi Soga, Takashi Yamamoto, Knut Woltjen
Gene-edited induced pluripotent stem cells (iPSCs) provide relevant isogenic human disease models in patient-specific or healthy genetic backgrounds. Towards this end, gene targeting using antibiotic selection along with engineered point mutations remains a reliable method to enrich edited cells. Nevertheless, integrated selection markers obstruct scarless transgene-free gene editing. Here, we present a method for scarless selection marker excision using engineered microhomology-mediated end joining (MMEJ)...
March 5, 2018: Nature Communications
https://www.readbyqxmd.com/read/29492861/analysis-of-housekeeping-genes-for-accurate-normalization-of-qpcr-data-during-early-postnatal-brain-development
#5
V A Shaydurov, A Kasianov, A P Bolshakov
Maturation of the neocortex during the first three postnatal weeks is a complex process that is characterized by different time courses of maturation of different areas of the neocortex. Analysis of gene expression using quantitative PCR after reverse transcription during this period of ontogeny and comparison of different cortical areas require optimal selection of reference genes for correct normalization of the data. Here, we compared expression of nine reference genes in the somatosensory and visual areas of the neocortex at the age of 5, 8, 10, 13, and 20 days...
February 28, 2018: Journal of Molecular Neuroscience: MN
https://www.readbyqxmd.com/read/29490947/metabolic-determinants-of-sensitivity-to-phosphatidylinositol-3-kinase-pathway-inhibitor-in-small-cell-lung-carcinoma
#6
Hideki Makinoshima, Shigeki Umemura, Ayako Suzuki, Hiroki Nakanishi, Ami Maruyama, Hibiki Udagawa, Sachiyo Mimaki, Shingo Matsumoto, Seiji Niho, Genichiro Ishii, Masahiro Tsuboi, Atsushi Ochiai, Hiroyasu Esumi, Takehiko Sasaki, Koichi Goto, Katsuya Tsuchihara
Comprehensive genomic analysis has revealed that the PI3K/AKT/mTOR pathway is a feasible therapeutic target in small-cell lung carcinoma (SCLC). However, biomarkers to identify patients likely to benefit from inhibitors of this pathway have not been identified. Here we show that metabolic features determine sensitivity to the PI3K/mTOR dual inhibitor gedatolisib in SCLC cells. Substantial phosphatidyl lipid analysis revealed that a specific phosphatidylinositol (3,4,5)-trisphosphate (PIP3) subspecies lipid product: PIP3 (38:4) is predictive in assessing sensitivity to PI3K/mTOR dual inhibitor...
February 28, 2018: Cancer Research
https://www.readbyqxmd.com/read/29479043/sound-exposure-induced-cytokine-gene-transcript-profile-changes-in-captive-bottlenose-dolphin-tursiops-truncatus-blood-identified-by-a-probe-based-qrt-pcr
#7
I-Hua Chen, Lien-Siang Chou, Shih-Jen Chou, Jiann-Hsiung Wang, Jeffrey Stott, Myra Blanchard, I-Fan Jen, Wei-Cheng Yang
Cetacean health may be potentially affected by anthropogenic sound. We have initiated investigations on the effect of low-frequency underwater sound on immunological gene transcript profiles of captive bottlenose dolphins (Tursiops truncatus) using a probe-based quantitative gene expression assay. Six immunologic genes (IL-2Rα, -4, -10, -12, TNFα, and IFNγ) were selected for analysis using two validated housekeeping genes (PGK1 and HPRT1) as reference genes. Twenty-four blood samples from six clinically healthy individuals and six blood samples from individuals after sound exposures were available...
February 26, 2018: Journal of Veterinary Medical Science
https://www.readbyqxmd.com/read/29474985/optimal-reference-gene-selection-for-expression-studies-in-human-reticulocytes
#8
Anu Aggarwal, Manu Jamwal, Ganesh Kumar V, Prashant Sharma, Man Updesh Singh Sachdeva, Deepak Bansal, Pankaj Malhotra, Reena Das
Reference genes are indispensable for normalizing mRNA levels across samples in real-time quantitative PCR. Their expression levels vary under different experimental conditions and due to several inherent characteristics. Appropriate reference gene selection is thus critical for gene-expression studies. This study aimed at selecting optimal reference genes for gene-expression analysis of reticulocytes and validating them in hereditary spherocytosis (HS) and β-thalassemia intermedia (βTI) patients. Seven reference genes (PGK1, MPP1, HPRT1, ACTB, GAPDH, RN18S1, and SDHA) were selected from published literature...
February 20, 2018: Journal of Molecular Diagnostics: JMD
https://www.readbyqxmd.com/read/29423928/unexpected-heterogeneity-derived-from-cas9-ribonucleoprotein-introduced-clonal-cells-at-the-hprt1-locus
#9
Tetsushi Sakuma, Keiji Mochida, Shota Nakade, Toru Ezure, Sachi Minagawa, Takashi Yamamoto
Single-cell cloning is an essential technique for establishing genome-edited cell clones mediated by programmable nucleases such as CRISPR-Cas9. However, residual genome-editing activity after single-cell cloning may cause heterogeneity in the clonal cells. Previous studies showed efficient mutagenesis and rapid degradation of CRISPR-Cas9 components in cultured cells by introducing Cas9 ribonucleoproteins (RNPs). In this study, we investigated how the timing for single-cell cloning of Cas9 RNP-transfected cells affected the heterogeneity of the resultant clones...
February 9, 2018: Genes to Cells: Devoted to Molecular & Cellular Mechanisms
https://www.readbyqxmd.com/read/29416770/hprt1-activity-loss-is-associated-with-resistance-to-thiopurine-in-all
#10
Fan Yang, Houshun Fang, Dan Wang, Yao Chen, Yonggong Zhai, Bin-Bing S Zhou, Hui Li
Acute lymphoblastic leukemia (ALL) is an aggressive hematological tumor resulting from the malignant transformation of lymphoid progenitors. Thiopurine is a widely used drug in the maintaining treatment of ALL. After a period of chemotherapy, 20% of pediatric patients and over 50% of adult patients will relapse. To investigate the mechanisms of drug resistance in vitro , we established the thiopurine resistant cell lines Reh-6MPR (6-MP Resistant cell) and Reh-6TGR (6-TG Resistant cell) by stepwise selection of the ALL cell line Reh...
January 5, 2018: Oncotarget
https://www.readbyqxmd.com/read/29386530/identification-of-valid-reference-genes-for-mrna-and-microrna-normalisation-in-prostate-cancer-cell-lines
#11
Hui Zhao, Teng-Fei Ma, Jie Lin, Lin-Lin Liu, Wei-Jie Sun, Li-Xia Guo, Si-Qi Wang, Newton O Otecko, Ya-Ping Zhang
RT-qPCR offers high sensitivity, for accurate interpretations of qPCR results however, normalisation using suitable reference genes is fundamental. Androgens can regulate transcriptional expression including reference gene expression in prostate cancer. In this study, we evaluated ten mRNA and six non-protein coding RNA reference genes in five prostate cell lines under varied dihydrotestosterone (DHT) treatments. We validated the effects of DHT-treatments using media containing charcoal-stripped serum prior to DHT stimulation on the test samples by Western blot experiments...
January 31, 2018: Scientific Reports
https://www.readbyqxmd.com/read/29219623/usual-normalization-strategies-for-gene-expression-studies-impair-the-detection-and-analysis-of-circadian-patterns
#12
Diego de Siqueira Figueredo, Mayara Rodrigues Barbosa, Daniel Gomes Coimbra, José Luiz Araújo Dos Santos, Ellyda Fernanda Lopes Costa, Bruna Del Vechio Koike, Magna Suzana Alexandre Moreira, Tiago Gomes de Andrade
Recent studies have shown that transcriptomes from different tissues present circadian oscillations. Therefore, the endogenous variation of total RNA should be considered as a potential bias in circadian studies of gene expression. However, normalization strategies generally include the equalization of total RNA concentration between samples prior to cDNA synthesis. Moreover, endogenous housekeeping genes (HKGs) frequently used for data normalization may exhibit circadian variation and distort experimental results if not detected or considered...
December 8, 2017: Chronobiology International
https://www.readbyqxmd.com/read/29209038/a-simple-method-to-assess-group-difference-in-rt-qpcr-reference-gene-selection-using-genorm-the-case-of-the-placental-sex
#13
Joey St-Pierre, Jean-Charles Grégoire, Cathy Vaillancourt
Normalization with proper reference genes is a crucial step in obtaining accurate mRNA expression levels in RT-qPCR experiments. GeNorm and NormFinder are two commonly used software packages that help in selecting the best reference genes, based on their expression stability. However, GeNorm does not take into account a group variable, such as sample sex, in its calculation. We demonstrate a simple calculation step to assess the variability of such parameters by multiplying the GeNorm M value with the difference of Cq values between groups...
December 5, 2017: Scientific Reports
https://www.readbyqxmd.com/read/29185864/novel-mutation-in-the-human-hprt1-gene-and-the-lesch-nyhan-disease
#14
Khue Vu Nguyen, Robert K Naviaux, William L Nyhan
Lesch-Nyhan disease (LND) is a rare X-linked inherited neurogenetic disorder of purine metabolism in which the enzyme, hypoxanthine-guanine phosphoribosyltransferase (HGprt) is defective. The authors report a novel point mutation that led to HGprt-related neurological dysfunction (HND) in a family in which there was a missense mutation in exon 6 of the coding region of the HPRT1 gene: g.34938G>T, c.403G>T, p.D135Y. Molecular diagnosis is consistent with the genetic heterogeneity of the HPRT1 gene responsible for HGprt deficiency...
November 2, 2017: Nucleosides, Nucleotides & Nucleic Acids
https://www.readbyqxmd.com/read/29180379/selection-of-internal-references-for-qrt-pcr-assays-of-human-hepatocellular-carcinoma-cell-lines
#15
Yang Liu, Zhaoyu Qin, Lili Cai, Lili Zou, Jing Zhao, Fan Zhong
Selecting internal references is important for normalizing the loading quantity of samples in quantitative reverse-transcription PCR (qRT-PCR). In the present study, a systematic evaluation of reference genes among nine hepatocellular carcinoma (HCC) cell lines was conducted. After screening the microarray assay data of ten HCC cell lines, 19 candidate reference genes were preselected and then evaluated by qRT-PCR, together with ACTB, GAPDH, HPRT1 and TUBB The expression evenness of these candidate genes was evaluated using RefFinder...
December 22, 2017: Bioscience Reports
https://www.readbyqxmd.com/read/29173213/evaluation-of-reference-genes-for-reverse-transcription-quantitative-pcr-assays-in-organs-of-zebrafish-exposed-to-glyphosate-based-herbicide-roundup
#16
M L Jaramillo, A G Pereira, C E Davico, L Nezzi, D Ammar, Y M R Müller, E M Nazari
Roundup is a glyphosate-based herbicide (GBH) widely used in agriculture and may cause toxic effects in non-target organisms. Model organisms, as zebrafish, and analysis of gene expression by reverse transcription-quantitative PCR (RT-qPCR) could be used to better understand the Roundup toxicity. A prerequisite for RT-qPCR is the availability of appropriate reference genes; however, they have not been described for Roundup-exposed fish. The aim of this study was to evaluate the expression stability of six reference genes (rpl8, β-act, gapdh, b2m, ef1α, hprt1) and one expressed repetitive element (hatn10) in organs of males (brain, gill, testis) and females (ovary) of zebrafish exposed to Roundup WG at three concentrations (0...
November 27, 2017: Animal: An International Journal of Animal Bioscience
https://www.readbyqxmd.com/read/29134774/identifying-suitable-reference-genes-for-developing-and-injured-mouse-cns-tissues
#17
Dongchao Xu, Ajuan Liu, Xuan Wang, Ming Zhang, Zunyi Zhang, Zhou Tan, Mengsheng Qiu
Accurate quantification of gene expression is fundamental for understanding the molecular, genetic and functional bases of tissue development and diseases. Quantitative real-time PCR (qPCR) is now the most widely used method of quantifying gene expression due to its simplicity, specificity, sensitivity, and wide quantification range. The use of appropriate reference genes to ensure accurate normalization is crucial for the correct quantification of gene expression from the early development, maturation, aging to injury processes in the central nervous system (CNS)...
January 2018: Developmental Neurobiology
https://www.readbyqxmd.com/read/29122836/multiplex-gene-expression-profiling-of-in-vivo-isolated-circulating-tumor-cells-in-high-risk-prostate-cancer-patients
#18
Athina Markou, Marifili Lazaridou, Panagiotis Paraskevopoulos, Shukun Chen, Monika Świerczewska, Joanna Budna, Andra Kuske, Tobias M Gorges, Simon A Joosse, Thomas Kroneis, Maciej Zabel, Peter Sedlmayr, Catherine Alix-Panabières, Klaus Pantel, Evi S Lianidou
BACKGROUND: Molecular characterization of circulating tumor cells (CTCs) is important for selecting patients for targeted treatments. We present, for the first time, results on gene expression profiling of CTCs isolated in vivo from high-risk prostate cancer (PCa) patients compared with CTC detected by 3 protein-based assays-CellSearch®, PSA-EPISPOT, and immunofluorescence of CellCollector® in vivo-captured CTCs-using the same blood draw. METHODS: EpCAM-positive CTCs were isolated in vivo using the CellCollector from 108 high-risk PCa patients and 36 healthy volunteers...
November 9, 2017: Clinical Chemistry
https://www.readbyqxmd.com/read/29115578/characterization-of-prostate-cancer-cell-progression-in-zebrafish-xenograft-model
#19
Wei Xu, Brittany A Foster, Mackenzie Richards, Kenneth R Bondioli, Girish Shah, Christopher C Green
Early diagnosis of prostate cancer (PCa) is critical for the application of efficient treatment to PCa patients. However, the majority of PCas remains indolent from several months to several years before malignancy. Current diagnosis methods have limitations in their reliability and are inefficient in time cost. Thus, an efficient in vivo PCa cell xenograft model is highly desired for diagnostic studies in PCas. In the present study we present a standardized procedure to create a PCa cell xenograft model using zebrafish (Danio rerio) as the host...
January 2018: International Journal of Oncology
https://www.readbyqxmd.com/read/29095075/suitable-reference-gene-selection-for-gene-expression-studies-in-knee-osteoarthritis-synovium-using-quantitative-pcr-analysis
#20
Hiroshi Watanabe, Hirotaka Ishii, Kenji Takahashi, Shinro Takai, Hitoshi Ozawa
Osteoarthritis (OA) is the leading cause of musculoskeletal disability in the elderly. Insights into the biological features of OA are obtained by characterization of the molecular features by gene expression profiling using reverse transcription-quantitative PCR (RT-qPCR). However, it has recently become evident that the use of suitable reference genes is required for appropriate normalization of this technique. Here total RNA was isolated from the synovium of 18 men and 20 women who underwent total knee arthroplasty for knee OA (KOA)...
November 2, 2017: Connective Tissue Research
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