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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
#1
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
#2
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 29, 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
#3
Yang Liu, Zhaoyu Qin, Lili Cai, Lili Zou, Jing Zhao, Fan Zhong
Selecting internal references is important for normalising the loading quantity of samples in quantitative reverse transcription polymerase chain reaction (qRT-PCR). In this study, a systematic evaluation of reference genes among nine hepatocellular carcinoma cell lines was conducted. After screening the microarray assay data of 10 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...
November 27, 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
#4
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
#5
Dongchao Xu, Ajuan Liu, Xuan Wang, Ming Zhang, Zunyi Zhang, Tan Zhou, 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)...
November 14, 2017: 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
#6
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
#7
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
#8
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
https://www.readbyqxmd.com/read/29053826/induction-of-somatic-mutations-by-low-dose-x-rays-the-challenge-in-recognizing-radiation-induced-events
#9
Haruki Nagashima, Kumiko Shiraishi, Saori Ohkawa, Yuki Sakamoto, Kenshi Komatsu, Shinya Matsuura, Akira Tachibana, Hiroshi Tauchi
It is difficult to distinguish radiation-induced events from spontaneous events during induction of stochastic effects, especially in the case of low-dose or low-dose-rate exposures. By using a hypersensitive system for detecting somatic mutations at the HPRT1 locus, we investigated the frequency and spectrum of mutations induced by low-dose X-rays. The mutant frequencies induced by doses of >0.15 Gy were statistically significant when compared with the spontaneous frequency, and a clear dose dependency was also observed for mutant frequencies at doses of >0...
October 19, 2017: Journal of Radiation Research
https://www.readbyqxmd.com/read/29036603/identification-of-optimal-reference-genes-for-transcriptomic-analyses-in-normal-and-diseased-human-heart
#10
Cristina E Molina, Eric Jacquet, Prishila Ponien, Christian Muñoz-Guijosa, Istvan Baczkó, Lars S Maier, Patrick Donzeau-Gouge, Dobromir Dobrev, Rodolphe Fischmeister, Anne Garnier
Aims: Quantitative real-time RT-PCR (RT-qPCR) has become the method of choice for mRNA quantification, but requires an accurate normalization based on the use of reference genes showing invariant expression across various pathological conditions. Only few data exist on appropriate reference genes for the human heart. The objective of this study was to determine a set of suitable reference genes in human atrial and ventricular tissues, from right and left cavities in control and in cardiac diseases...
September 22, 2017: Cardiovascular Research
https://www.readbyqxmd.com/read/28949053/cervical-cancer-stem-like-cells-systematic-review-and-identification-of-reference-genes-for-gene-expression
#11
Rafael Paschoal de Campos, Iago Carvalho Schultz, Paola de Andrade Mello, Samuel Davies, Manuela Sangalli Gasparin, Ana Paula Santin Bertoni, Andréia Buffon, Márcia Rosângela Wink
Cervical cancer is the fourth most common cancer affecting women worldwide. Among many factors, the presence of cancer stem cells, a subpopulation of cells inside the tumor, has been associated with a worse prognosis. Considering the importance of gene expression studies to understand the biology of cervical cancer stem cells (CCSC), this work identifies stable reference genes for cervical cancer cell lines SiHa, HeLa and ME180 as well as their respective cancer stem-like cells. A literature review was performed to identify validated reference genes currently used to normalize RT-qPCR data in cervical cancer cell lines...
September 26, 2017: Cell Biology International
https://www.readbyqxmd.com/read/28880574/selection-of-suitable-internal-control-genes-for-accurate-normalization-of-real-time-quantitative-pcr-data-of-buffalo-bubalus-bubalis-blastocysts-produced-by-scnt-and-ivf
#12
Tanushri Jerath Sood, Swati Viviyan Lagah, Ankita Sharma, Suresh Kumar Singla, Manishi Mukesh, Manmohan Singh Chauhan, Radheysham Manik, Prabhat Palta
We evaluated the suitability of 10 candidate internal control genes (ICGs), belonging to different functional classes, namely ACTB, EEF1A1, GAPDH, HPRT1, HMBS, RPS15, RPS18, RPS23, SDHA, and UBC for normalizing the real-time quantitative polymerase chain reaction (qPCR) data of blastocyst-stage buffalo embryos produced by hand-made cloning and in vitro fertilization (IVF). Total RNA was isolated from three pools, each of cloned and IVF blastocysts (n = 50/pool) for cDNA synthesis. Two different statistical algorithms geNorm and NormFinder were used for evaluating the stability of these genes...
October 2017: Cellular Reprogramming
https://www.readbyqxmd.com/read/28868355/stability-of-gene-expression-in-human-t-cells-in-different-gravity-environments-is-clustered-in-chromosomal-region-11p15-4
#13
Cora S Thiel, Andreas Huge, Swantje Hauschild, Svantje Tauber, Beatrice A Lauber, Jennifer Polzer, Katrin Paulsen, Hartwin Lier, Frank Engelmann, Burkhard Schmitz, Andreas Schütte, Liliana E Layer, Oliver Ullrich
In the last decades, a plethora of in vitro studies with living human cells contributed a vast amount of knowledge about cellular and molecular effects of microgravity. Previous studies focused mostly on the identification of gravity-responsive genes, whereas a multi-platform analysis at an integrative level, which specifically evaluates the extent and robustness of transcriptional response to an altered gravity environment was not performed so far. Therefore, we investigated the stability of gene expression response in non-activated human Jurkat T lymphocytic cells in different gravity environments through the combination of parabolic flights with a suborbital ballistic rocket and 2D clinostat and centrifuge experiments, using strict controls for excluding all possible other factors of influence...
2017: NPJ Microgravity
https://www.readbyqxmd.com/read/28861591/multianalyte-quantitative-competitive-pcr-on-optically-encoded-microspheres-for-an-eight-gene-panel-related-to-prostate-cancer
#14
Iraklis K Kyriakou, Konstantinos Mavridis, Despina P Kalogianni, Theodore K Christopoulos, Penelope C Ioannou, Andreas Scorilas
Nucleic acid-based tests have a profound impact in every medical discipline. Because multigene tests offer higher diagnostic accuracy and lower overall cost than single assays, they are especially useful for diseases, like prostate cancer, that present variability at the molecular level and diversity of available therapeutic interventions. We have developed a quantitative competitive PCR for an eight-gene panel, related to prostate cancer, that includes five genes of the human tissue kallikrein family (KLKs), prostate-specific membrane antigen (PSMA), prostate cancer antigen 3 (PCA3), and HPRT1 as a reference gene...
August 31, 2017: Analytical and Bioanalytical Chemistry
https://www.readbyqxmd.com/read/28782500/unapparent-hypoxanthine-guanine-phosphoribosyltransferase-deficiency
#15
R J Torres, S Puente, A Menendez, N Fernandez-Garcia
Complete deficiency of hypoxanthine-guanine phosphoribosyltransferase (HPRT) activity causes Lesch Nyhan disease (LND), characterized by hyperuricemia, severe action dystonia, choreoathetosis, ballismus, cognitive and attention deficit and self-injurious behavior. Partial HPRT deficiency is present in patients with Lesch-Nyhan variant (LNV), who present with HPRT-related gout and a variable degree of neurological involvement. The diagnosis of HPRT deficiency relies on clinical, biochemical, enzymatic and molecular data...
September 2017: Clinica Chimica Acta; International Journal of Clinical Chemistry
https://www.readbyqxmd.com/read/28761164/identification-of-reference-genes-for-qrt-pcr-in-granulosa-cells-of-healthy-women-and-polycystic-ovarian-syndrome-patients
#16
Yue Lv, Shi Gang Zhao, Gang Lu, Chi Kwan Leung, Zhi Qiang Xiong, Xian Wei Su, Jin Long Ma, Wai Yee Chan, Hong Bin Liu
Comparative gene expression analysis by qRT-PCR is commonly used to detect differentially expressed genes in studies of PCOS pathology. Impaired GC function is strongly associated with PCOS pathogenesis, and a growing body of studies has been dedicated to identifying differentially expressed genes in GCs in PCOS patients and healthy women by qRT-PCR. It is necessary to validate the expression stability of the selected reference genes across the tested samples for target gene expression normalization. We examined the variability and stability of expression of the 15 commonly used reference genes in GCs from 44 PCOS patients and 45 healthy women using the GeNorm, BestKeeper, and NormFinder statistical algorithms...
July 31, 2017: Scientific Reports
https://www.readbyqxmd.com/read/28732075/comprehensive-selection-of-reference-genes-for-quantitative-rt-pcr-analysis-of-murine-extramedullary-hematopoiesis-during-development
#17
Giuliana Medrano, Peihong Guan, Amanda J Barlow-Anacker, Ankush Gosain
The purpose of this study was to perform a comprehensive evaluation and selection of reference genes for the study of extramedullary hematopoiesis during development and the early post-natal period. A total of six candidate reference genes (ACTB, GAPDH, HPRT1, PPID, TBP, TUBB3) in four organs (heart, liver, spleen, and thymus) over five perinatal time points (Embryonic days 14.5, 16.5, 18.5, Post-natal days 0, 21) were evaluated by quantitative real-time PCR. The expression stability of the candidate reference genes were analyzed using geNorm, NormFinder, Bestkeeper, Delta CT method, and RefFinder software packages...
2017: PloS One
https://www.readbyqxmd.com/read/28712454/crispr-cas9-mediated-scanning-for-regulatory-elements-required-for-hprt1-expression-via-thousands-of-large-programmed-genomic-deletions
#18
Molly Gasperini, Gregory M Findlay, Aaron McKenna, Jennifer H Milbank, Choli Lee, Melissa D Zhang, Darren A Cusanovich, Jay Shendure
The extent to which non-coding mutations contribute to Mendelian disease is a major unknown in human genetics. Relatedly, the vast majority of candidate regulatory elements have yet to be functionally validated. Here, we describe a CRISPR-based system that uses pairs of guide RNAs (gRNAs) to program thousands of kilobase-scale deletions that deeply scan across a targeted region in a tiling fashion ("ScanDel"). We applied ScanDel to HPRT1, the housekeeping gene underlying Lesch-Nyhan syndrome, an X-linked recessive disorder...
August 3, 2017: American Journal of Human Genetics
https://www.readbyqxmd.com/read/28671969/reference-gene-selection-for-the-shell-gland-of-laying-hens-in-response-to-time-points-of-eggshell-formation-and-nicarbazin
#19
Sami Samiullah, Juliet Roberts, Shu-Biao Wu
Ten reference genes were investigated for normalization of gene expression data in the shell gland of laying hens. Analyses performed with geNorm revealed that hypoxanthine phosphoribosyltransferase 1 (HPRT1) and hydroxymethylbilane synthase (HMBS) were the two most stable reference genes in response to post-oviposition time alone (POT) or with nicarbazin treatment (POT+N) of laying hens. NormFinder analyses showed that the two most stable reference genes in response to POT and POT+N were 18S ribosomal RNA (18S rRNA), ribosomal protein L4 (RPL4) and HMBS, RPL4, respectively...
2017: PloS One
https://www.readbyqxmd.com/read/28524722/lesch-nyhan-disease-in-two-families-from-chilo%C3%A3-island-with-mutations-in-the-hprt1-gene
#20
Khue Vu Nguyen, Sebastian Silva, Monica Troncoso, 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 two independent point mutations leading to splicing errors: IVS 2 +1G>A, c.134 +1G>A, and IVS 3 +1G>A, c.318 +1G>A in the hypoxanthine-phosphoribosyltransferase1 (HPRT1) gene which result in exclusion of exon 2 and exon 3 respectively, in the HGprt enzyme protein from different members of two Chiloé Island families...
July 3, 2017: Nucleosides, Nucleotides & Nucleic Acids
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