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https://www.readbyqxmd.com/read/25743365/evaluation-of-potential-internal-references-for-quantitative-real-time-rt-pcr-normalization-of-gene-expression-in-red-drum-sciaenops-ocellatus
#1
Bo-Guang Sun, Yong-Hua Hu
Quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR) has been used extensively for studying gene expression in diverse organisms including fish. In this study, with an aim to identify reliable reference genes for qRT-PCR in red drum (Sciaenops ocellatus), an economic fish species, we determined the expression stability of seven housekeeping genes in healthy and bacterium-infected red drum. Each of the selected candidate genes was amplified by qRT-PCR from the brain, gill, heart, intestine, kidney, liver, muscle, and spleen of red drum infected with or without a bacterial pathogen for 12 and 48 h...
June 2015: Fish Physiology and Biochemistry
https://www.readbyqxmd.com/read/25550045/selection-of-appropriate-reference-genes-for-rt-qpcr-analysis-in-berkshire-duroc-landrace-and-yorkshire-pigs
#2
Sang-Je Park, Seul Gi Kwon, Jung Hye Hwang, Da Hye Park, Tae Wan Kim, Chul Wook Kim
Reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR) is the most reliable molecular biology technique for assessment of mRNA expression levels. However, to obtain the accurate RT-qPCR results, the expression levels of genes of interest should be normalized with appropriate reference genes and optimal numbers of reference genes. In this study, we assessed the expression stability of 15 well-known candidate reference genes (ACTB, ALDOA, B2M, GAPDH, HPAR1, HSPCB, PGK1, POLR2G, PPIA, RPL4, RPS18, SDHA, TBP, TOP2B, and YWHAZ) in seven body tissues (liver, lung, kidney, spleen, stomach, small intestine, and large intestine) of Berkshire, Landrace, Duroc, and Yorkshire pigs using three excel-based programs, geNorm, NormFinder, and BestKeeper...
March 1, 2015: Gene
https://www.readbyqxmd.com/read/25049806/selection-of-reliable-reference-genes-for-real-time-qrt-pcr-analysis-of-zi-geese-anser-anser-domestica-gene-expression
#3
Hong Ji, Jianfa Wang, Juxiong Liu, Jingru Guo, Zhongwei Wang, Xu Zhang, Li Guo, Huanmin Yang
UNLABELLED: Zi geese (Anser anser domestica) belong to the white geese and are excellent layers with a superior feed-to-egg conversion ratio. Quantitative gene expression analysis, such as Real-time qRT-PCR, will provide a good understanding of ovarian function during egg-laying and consequently improve egg production. However, we still don't know what reference genes in geese, which show stable expression, should be used for such quantitative analysis. In order to reveal such reference genes, the stability of seven genes were tested in five tissues of Zi geese...
March 2013: Asian-Australasian Journal of Animal Sciences
https://www.readbyqxmd.com/read/25049756/reference-gene-screening-for-analyzing-gene-expression-across-goat-tissue
#4
Yu Zhang, Xiao-Dong Zhang, Xing Liu, Yun-Sheng Li, Jian-Ping Ding, Xiao-Rong Zhang, Yun-Hai Zhang
Real-time quantitative PCR (qRT-PCR) is one of the important methods for investigating the changes in mRNA expression levels in cells and tissues. Selection of the proper reference genes is very important when calibrating the results of real-time quantitative PCR. Studies on the selection of reference genes in goat tissues are limited, despite the economic importance of their meat and dairy products. We used real-time quantitative PCR to detect the expression levels of eight reference gene candidates (18S, TBP, HMBS, YWHAZ, ACTB, HPRT1, GAPDH and EEF1A2) in ten tissues types sourced from Boer goats...
December 2013: Asian-Australasian Journal of Animal Sciences
https://www.readbyqxmd.com/read/24687048/renal-tissue-thawed-for-30-minutes-is-still-suitable-for-gene-expression-analysis
#5
Yi Ma, Xiao-Nan Kang, Wen-Bin Ding, Hao-Zheng Yang, Ye Wang, Jin Zhang, Yi-Ran Huang, Hui-Li Dai
Some biosamples obtained from biobanks may go through thawing before processing. We aim to evaluate the effects of thawing at room temperature for different time periods on gene expression analysis. A time course study with four time points was conducted to investigate the expression profiling on 10 thawed normal mice renal tissue samples through Affymetrix GeneChip mouse gene 2.0 st array. Microarray results were validated by quantitative real time polymerase chain reactions (qPCR) on 6 candidate reference genes and 11 target genes...
2014: PloS One
https://www.readbyqxmd.com/read/24368222/determination-of-reference-micrornas-for-relative-quantification-in-grass-carp-ctenopharyngodon-idella
#6
Xiao-Yan Xu, Yu-Bang Shen, Jian-Jun Fu, Li-Qun Lu, Jia-Le Li
Relative quantification is the strategy of choice for processing real-time quantitative reverse transcription polymerase chain reaction (RT-qPCR) data in microRNA (miRNA) expression studies. Normalization of relative quantification data is performed by comparison to reference genes. In teleost species, such as grass carp (Ctenopharyngodon idella), the determination of reference miRNAs and the optimal numbers of these that should be used has not been widely studied. In the present study, the stability of seven miRNAs (miR-126-3p, miR-101a, miR-451, miR-22a, miR-146, miR-142a-5p and miR-192) was investigated by RT-qPCR in different tissues and in different development stages of grass carp...
February 2014: Fish & Shellfish Immunology
https://www.readbyqxmd.com/read/23601718/the-selection-of-endogenous-genes-in-human-postmortem-tissues
#7
Heng Zhang, Ping Zhang, Kai-Jun Ma, Ye-Hui Lv, Wen-Can Li, Cheng-Liang Luo, Li-Liang Li, Yi-Wen Shen, Meng He, Jie-Qing Jiang, Long Chen
Precisely determining the postmortem interval (PMI), which is crucial to criminal and forensic cases, is a research in which quantitative RT-PCR (also known as qRT-PCR or real-time RT-PCR) has been used to analyse gene expression levels and data normalisation should be required to eliminate the differences among the samples. Therefore, it is quite necessary to find stable molecular biological markers in PMI determination research. In this study, we compared nine commonly used endogenous markers (containing ACTB, GAPDH, B2M, U6, 18S rRNA, hsa-mir-1, hsa-mir-9, hsa-mir-194-1 and hsa-mir-203) in the 109 human tissue samples obtained from autopsy at the aim of finding stable markers in human tissues with consideration of the impact of parameters (PMI and cause of death)...
June 2013: Science & Justice: Journal of the Forensic Science Society
https://www.readbyqxmd.com/read/23332581/selection-of-normalization-factors-for-quantitative-real-time-rt-pcr-studies-in-japanese-flounder-paralichthys-olivaceus-and-turbot-scophthalmus-maximus-under-conditions-of-viral-infection
#8
Jian Zhang, Yong-hua Hu, Bo-guang Sun, Zhi-zhong Xiao, Li Sun
Disease outbreaks caused by iridoviruses are known to affect farmed flounder (Paralichthys olivaceus) and turbot (Scophthalmus maximus). To facilitate quantitative real time RT-PCR (qRT-PCR) analysis of gene expression in flounder and turbot during viral infection, we in this study examined the potentials of 9 housekeeping genes of flounder and turbot as internal references for qRT-PCR under conditions of experimental infection with megalocytivirus, a member of the Iridoviridae family. The mRNA levels of the 9 housekeeping genes in the brain, gill, heart, intestine, kidney, liver, muscle, and spleen of flounder and turbot were determined by qRT-PCR at 24h and 72h post-viral infection, and the expression stabilities of the genes were determined with geNorm and NormFinder algorithms...
April 15, 2013: Veterinary Immunology and Immunopathology
https://www.readbyqxmd.com/read/23069845/ppar%C3%AE-an-important-gene-related-to-lipid-metabolism-and-immunity-in-megalobrama-amblycephala-cloning-characterization-and-transcription-analysis-by-genorm
#9
Su Li, Yasmeen Gul, Weimin Wang, Xueqiao Qian, Yuhua Zhao
In order to be able to modulate and improve the function of PPARγ and decrease further some metabolic diseases of M. amblycephala, we have cloned and identified the full-length cDNA of PPARγ in M. amblycephala and examined its transcription patterns at different embryo developmental stages and in different tissues of adult and immature fish. We also accurately normalized seven reference genes by GeNorm and calculated their gene transcription normalization factors. The full-length of PPARγ was 1968 bp, consisting of 218 bp 5'-untranslated region, 1,533 bp open reading frame encoding 510 amino acids residues and 217 bp 3'-untranslated region...
January 10, 2013: Gene
https://www.readbyqxmd.com/read/22970213/determination-of-reference-micrornas-for-relative-quantification-in-porcine-tissues
#10
Oriol Timoneda, Ingrid Balcells, Sarai Córdoba, Anna Castelló, Armand Sánchez
Relative quantification is the strategy of choice for processing RT-qPCR data in microRNAs (miRNAs) expression studies. Normalisation of relative quantification data is performed by using reference genes. In livestock species, such as pigs, the determination of reference miRNAs and the optimal number of them has not been widely studied. In this study, the stability of ten miRNAs (Ssc-let-7a, Ssc-miR-103, Ssc-miR-17-3p, Hsa-miR-25, Hsa-miR-93, Ssc-miR-106a, Ssc-miR-191, Ssc-miR-16, Ssc-miR-26a and Ssc-miR-17-5p) was investigated by RT-qPCR in different tissues (skeletal muscle, kidney, liver, ovary and uterus) and in different pig breeds (Iberian, Landrace, Large White, Meishan and Vietnamese) as variation factors...
2012: PloS One
https://www.readbyqxmd.com/read/22847160/impact-of-thawing-on-reference-gene-expression-stability-in-renal-cell-carcinoma-samples
#11
Yi Ma, HuiLi Dai, XianMing Kong, LiMin Wang
More and more samples are obtained from biobanks for biomedical research; however, some of these samples may undergo thawing before processing. We aim to evaluate the reference gene expression stability in thawed renal cell carcinoma samples. Sixteen matched malignant and nonmalignant renal tissue samples were obtained and each sample was divided into 4 aliquots before being snap frozen and stored at -80°C. By quantitative real-time polymerase chain reaction, a time-course study was conducted on the thawed tissue to evaluate the expression stability of a panel of the 10 most frequently used reference genes in renal cell carcinom samples: ACTB, ALAS1, B2M, GAPDH, HMBS, HPRT, PPIA, RPLP0,TBP, and TUBB...
September 2012: Diagnostic Molecular Pathology: the American Journal of Surgical Pathology, Part B
https://www.readbyqxmd.com/read/22788411/evaluation-of-reference-genes-for-the-analysis-of-serum-mirna-in-patients-with-prostate-cancer-bladder-cancer-and-renal-cell-carcinoma
#12
Imke Sanders, Stefan Holdenrieder, Gisela Walgenbach-Brünagel, Alexander von Ruecker, Glen Kristiansen, Stefan C Müller, Jörg Ellinger
OBJECTIVES: To identify an appropriate reference gene for the analysis of circulating micro-ribonucleic acid in patients with urological malignancies. METHODS: Serum from patients with prostate cancer (n = 24), bladder cancer (n = 24), renal cell carcinoma (n = 24) and control subjects (n = 48) was spiked with cel-miR-39, and then ribonucleic acid was isolated. Quantitative real-time polymerase chain reaction was used to determine the levels of candidate reference genes (RNU1-4, RNU6-2, SNORD43, SNORD44, SNORD48, SNORA74A, miR-let-7a-1, miR-106a)...
November 2012: International Journal of Urology: Official Journal of the Japanese Urological Association
https://www.readbyqxmd.com/read/21745577/cloning-identification-and-accurate-normalization-expression-analysis-of-ppar%C3%AE-gene-by-genorm-in-megalobrama-amblycephala
#13
Yuhua Zhao, Yasmeen Gul, Su Li, Weimin Wang
Megalobrama amblycephala suffers from serious liver diseases recently and PPARα gene has been reported to play an important role in the immune system of animal liver. On the basis of these facts, we have cloned and identified full-length cDNA of PPARα and examined its expression patterns at different embryo developmental stages and in different tissues of adult and young fish in order to improve liver disease immunity of M. amblycephala. We also accurately normalized seven reference genes by GeNorm and calculated their gene expression normalization factors...
September 2011: Fish & Shellfish Immunology
https://www.readbyqxmd.com/read/21741950/reference-genes-for-the-relative-quantification-of-micrornas-in-renal-cell-carcinomas-and-their-metastases
#14
Zofia Wotschofsky, Helmuth-Alexander Meyer, Monika Jung, Annika Fendler, Ina Wagner, Carsten Stephan, Jonas Busch, Andreas Erbersdobler, Alexander C Disch, Hans-Joachim Mollenkopf, Klaus Jung
To obtain accurate results in miRNA expression changes between different sample sets using real-time quantitative polymerase chain reaction (RT-qPCR) analyses, normalization to reference genes that are stably expressed across the sample sets is generally used. A literature search of miRNA expression studies in renal cell carcinoma (RCC) proved that non-miRNAs such as small RNAs or mRNAs have most frequently been used without preceding validation of their suitability. In this study, the most stably expressed miRNAs were ascertained from microarray-based data of miRNA expression in nonmalignant and malignant samples from clear cell RCC and from corresponding distant RCC metastases using the geNorm and NormFinder algorithms...
October 15, 2011: Analytical Biochemistry
https://www.readbyqxmd.com/read/21673918/evaluation-of-potential-reference-genes-for-relative-quantification-by-rt-qpcr-in-different-porcine-tissues-derived-from-feeding-studies
#15
Qimeng Li, Konrad Johann Domig, Thomas Ettle, Wilhelm Windisch, Christiane Mair, Karl Schedle
Five potential reference genes for RT-qPCR application, namely histone H3, beta-actin, GAPDH, ubiquitin and 18S rRNA, were evaluated for normalization of gene expression in four selected tissues (liver, kidney, thyroid and abdominal fat). Tissues were derived from fattening pigs exposed to different amounts and type of dietary iodine. Two software applications (geNorm and NormFinder) were used to evaluate the stability of the potential reference genes. All studied genes displayed high expression stability but different stability patterns between the investigated tissues...
2011: International Journal of Molecular Sciences
https://www.readbyqxmd.com/read/21255653/evaluation-of-internal-control-genes-for-qrt-pcr-normalization-in-tissues-and-cell-culture-for-antiviral-studies-of-grass-carp-ctenopharyngodon-idella
#16
Jianguo Su, Rongfang Zhang, Jie Dong, Chunrong Yang
Real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR) has become one of the most commonly used techniques for RNA expression. To obtain more reliable results with biological significance, it requires data normalization using an appropriate internal control gene. Here, we cloned partial sequence of elongation factor 1α (EF1α) gene from grass carp (Ctenopharyngodon idella). The stabilities of four commonly used internal control genes encoding 18S rRNA, β-actin, EF1α, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were integratedly assessed using the geNorm, NormFinder and BestKeeper programs...
March 2011: Fish & Shellfish Immunology
https://www.readbyqxmd.com/read/21220029/determination-of-internal-controls-for-quantitative-real-time-rt-pcr-analysis-of-the-effect-of-edwardsiella-tarda-infection-on-gene-expression-in-turbot-scophthalmus-maximus
#17
Wei Dang, Li Sun
In recent years, quantitative real time reverse transcriptase-PCR (qRT-PCR) has been used frequently in the study of gene expression in turbot (Scophthalmus maximus) in relation to bacterial infection. However, no investigations on appropriate qRT-PCR reference genes have been documented. In this report, we determined the potential of eight housekeeping genes, i.e. β-actin (ACTB), ribosomal protein L17 (RPL17), α-tubulin (TUBA), elongation factor-1-α(EF1A), β-2-Microglobulin (B2M), RNA polymerase II subunit D (RPSD), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and 18S ribosomal RNA (18S rRNA), as internal standards for qRT-PCR analysis of gene expression in turbot as a function of bacterial infection...
February 2011: Fish & Shellfish Immunology
https://www.readbyqxmd.com/read/21185941/evaluation-of-housekeeping-genes-as-references-for-quantitative-real-time-rt-pcr-analysis-of-gene-expression-in-japanese-flounder-paralichthys-olivaceus
#18
Wen-Jiang Zheng, Li Sun
Japanese flounder (Paralichthys olivaceus) is an important economic fish species cultured worldwide. In this report, we compared the potentials of ten housekeeping genes as quantitative real time RT-PCR (qRT-PCR) references for the study of gene expression in Japanese flounder under normal physiological conditions and during bacterial infection. For this purpose, the expression of the ten genes in eight flounder tissues (liver, spleen, kidney, heart, muscle, brain, gill, and intestine) was determined by qRT-PCR before and after bacterial infection...
February 2011: Fish & Shellfish Immunology
https://www.readbyqxmd.com/read/20459764/evaluation-of-potential-reference-genes-for-real-time-rt-pcr-studies-in-atlantic-halibut-hippoglossus-hippoglossus-l-during-development-in-tissues-of-healthy-and-nnv-injected-fish-and-in-anterior-kidney-leucocytes
#19
Aina-Cathrine Øvergård, Audun Helge Nerland, Sonal Patel
BACKGROUND: Real time RT-PCR has become an important tool for analyzing gene expression in fish. Although several housekeeping genes have been evaluated in Atlantic halibut (Hippoglossus Hippoglossus L.), appropriate reference genes for low copy mRNA transcripts at the earliest developmental stages have not been identified. No attempts have been reported to identify suitable reference genes in halibut infected with NNV or in stimulated halibut leucocytes. In this study, beta-actin1 (ACTB1), elongation factor 1 alpha (EF1A1), hypoxanthine-guanine phosphoribosyltransferase 1 (HPRT1), ribosomal protein L7 (RPL7), tubulin beta 2C (Tubb2C), and ubiquitin-conjugating enzyme (UbcE) were evaluated as reference genes for normalization of real time RT-PCR data during Atlantic halibut development, in tissue of healthy and NNV-infected fish, and in in vivo and in vitro stimulated anterior kidney leucocytes...
2010: BMC Molecular Biology
https://www.readbyqxmd.com/read/19638286/reference-genes-evaluated-for-use-in-infectious-pancreatic-necrosis-virus-real-time-rt-qpcr-assay-applied-during-different-stages-of-an-infection
#20
K Julin, L-H Johansen, A-I Sommer
The stability of 6 reference genes, 18S, beta-actin, RPS20, eEF1alpha, G6PDH and GAPDH, was examined in tissues from Atlantic salmon (Salmo salar) and Chinook salmon embryo cells (CHSE-214). The main objective of this study was to determine the most suitable reference genes for use for the normalisation of data in quantitative real-time RT-qPCR assays conducted on infected tissues. The tissue samples selected for analysis were taken from head kidney and pylorus and collected at different time points during a challenge experiment with infectious pancreatic necrosis virus (IPNV)...
December 2009: Journal of Virological Methods
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