scholarly journals A set of endogenous control genes for use in quantitative real-time PCR experiments reveal that the wild-type formin Ldia2 is enriched in the early pond snail embryo

2019 ◽  
Author(s):  
Harriet F. Johnson ◽  
Angus Davison
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Lymnaea Stagnalis ◽  
Model Organism ◽  
Early Embryo ◽  
Pond Snail ◽  
Endogenous Control ◽  
Wild Type ◽  
Quantitative Real Time Pcr ◽  
Nonsense Mediated Decay

ABSTRACTAlthough the pond snail Lymnaea stagnalis is an emerging model organism for molecular studies in a wide variety of fields including development, biomineralisation and neurophysiology, there are a limited number of verified endogenous control genes for use in quantitative real-time PCR (qRT-PCR). As part of larger study on snail chirality or left-right asymmetry, we wished to assay relative gene expression in pond snail embryos, so we evaluated six new candidate control genes, by comparing their expression in three tissues (ovotestis, foot, and embryo) and across three programs (geNorm, Normfinder and Bestkeeper). The specific utility of these control genes was then tested by investigating the relative expression of six experimental transcripts, including the formin Ldia2, a gene that has been associated with chirality in L. stagnalis. All six control genes were found to be suitable for use. Of the six experimental genes that were tested, it was found that all were relatively depleted in the early embryo compared with other tissues, except the formin gene Ldia2. Instead, transcripts of the wild type Ldia2dex were enriched in the embryo, whereas a non-functional frameshifted version Ldia2sin was severely depleted. These differences in Ldia2sin expression were less evident in the ovotestis and not evident in the foot tissue, suggesting that nonsense-mediated decay may be obscured in actively transcribing tissues. This work therefore provides a set of control genes that may be useful to the wider community, and shows how they may be used to assay differences in expression in the early embryo.

scholarly journals A new set of endogenous control genes for use in quantitative real-time PCR experiments show that formin Ldia2dex transcripts are enriched in the early embryo of the pond snail Lymnaea stagnalis (Panpulmonata)

2019 ◽  
Author(s):  
Harriet F Johnson ◽  
Angus Davison
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Lymnaea Stagnalis ◽  
Model Organism ◽  
Early Embryo ◽  
Pond Snail ◽  
Endogenous Control ◽  
Quantitative Real Time Pcr ◽  
Nonsense Mediated Decay ◽  
Left Right Asymmetry

ABSTRACT Although the pond snail Lymnaea stagnalis is an emerging model organism for molecular studies in a wide variety of fields, there are a limited number of verified endogenous control genes for use in quantitative real-time PCR. As part of a larger study on snail chirality, or left–right asymmetry, we assayed gene expression in pond snail embryos. We evaluated six candidate control genes, by comparing their expression in three tissues (ovotestis, foot and embryo) and used three software programmes (geNorm, Normfinder and Bestkeeper) to do so. The specific utility of these control genes was then tested by investigating the relative expression of six experimental transcripts, including formin Ldia2, a gene that has been associated with chiral variation in L. stagnalis. All six control genes were found to be suitable for use in the three tissues tested. Of the six experimental genes, it was found that all were relatively depleted in the early embryo compared with other tissues, except the formin Ldia2 gene. Instead, transcripts of the wild-type Ldia2dex were enriched in the embryo, whereas a nonfunctional frameshifted version, Ldia2sin, was severely depleted. These differences in Ldia2sin expression were less evident in the ovotestis and were not evident in the foot tissue, possibly because nonsense-mediated decay is obscured in actively transcribing tissues. Our work provides a set of control genes that may be useful to the wider community and illustrates how these genes may be used to assay differences in expression in a variety of tissues.

scholarly journals Comprehensive Human Adipose Tissue mRNA and MicroRNA Endogenous Control Selection for Quantitative Real-Time-PCR Normalization

Obesity ◽  
2011 ◽  
Vol 19 (4) ◽  
pp. 888-892 ◽  
Author(s):  
Matt J. Neville ◽  
Jenny M. Collins ◽  
Anna L. Gloyn ◽  
Mark I. McCarthy ◽  
Fredrik Karpe
Keyword(s):  
Adipose Tissue ◽  
Real Time ◽  
Real Time Pcr ◽  
Human Adipose Tissue ◽  
Endogenous Control ◽  
Quantitative Real Time Pcr ◽  
Control Selection ◽  
Selection For

scholarly journals Anti-influenza A virus activity of two Newtonia species and the isolated compound myricetin-3-o-rhamnoside

2021 ◽  
Vol 21 (1) ◽  
Author(s):  
Katlego E. Motlhatlego ◽  
Parvaneh Mehrbod ◽  
Fatemeh Fotouhi ◽  
Muna Ali Abdalla ◽  
Jacobus N. Eloff ◽  
...  
Keyword(s):  
Real Time ◽  
Influenza A Virus ◽  
Real Time Pcr ◽  
Influenza A ◽  
Combined Treatment ◽  
Model Organism ◽  
Quantitative Real Time Pcr ◽  
Leaf Extracts ◽  
Viral Attachment ◽  
Isolated Compound

Abstract Background Some viruses play a key role in the disturbance of the digestive system. The common viruses which cause infectious diarrhoea (gastroenteritis) include astrovirus, caliciviruses, coronavirus and torovirus which are single-stranded RNA viruses. Influenza A virus (H1N1) also causes diarrhoea in addition to being associated with respiratory symptoms. In preliminary studies, Newtonia hildebrandtii and N. buchananii leaf extracts had good antibacterial activity against some bacteria implicated in causing diarrhoea. The aim of this study was to evaluate the anti-influenza activity of two Newtonia species extracts and the isolated compound (myricitrin). Methods N. hildebrandtii and N. buchananii acetone, and MeOH: DCM (methanol-dichloromethane) leaf and stem extracts, and an antibacterial compound myricetin-3-o-rhamnoside (myricitrin), isolated from N. buchananii, were evaluated for their antiviral efficacy against influenza A virus (IAV) PR8/34/H1N1 as a model organism. The MTT and hemagglutination assays were used to assess the extracts and compound interference with cell viability and viral surface HA glycoprotein. The quantitative real-time PCR was performed to assess the viral load. Results Plant extracts of N. hildebrandtii and N. buchananii were effective against IAV. The extracts in combination with H1N1 showed highly significant antiviral activity (P < 0.01) and maintained cell viabilities (P < 0.05). Myricitrin was non-cytotoxic at concentration 104 μg/ml. Myricitrin was most effective against IAV in a co-penetration combined treatment, thereby confirming the inhibitory effect of this compound in the viral attachment and entry stages. Myricitrin treatment also resulted in the highest viability of the cells in co-penetration treatment. The activity of myricitrin indicates the potential of the extracts in controlling viral infection at the attachment stage. The antiviral effect of myricitrin on IAV load in MDCK cell culture was confirmed using quantitative real-time PCR. Conclusion Data from this study support further research and development on Newtonia hildebrandtii, Newtonia buchananii and myricitrin to address diarrhoea and related conditions caused by viruses in both human and veterinary medicine. Further work needs to be conducted on the activity of the extracts and the purified compound on other viruses of importance which have similar symptoms to influenza virus such as the coronavirus which led to a recent global pandemic.

scholarly journals Rapid method for identification of transgenic fish zygosity

2007 ◽  
Vol 6 (2) ◽  
pp. 177
Author(s):  
. Alimuddin ◽  
G. Yoshizaki ◽  
O. Carman
Keyword(s):  
Real Time ◽  
Danio Rerio ◽  
Real Time Pcr ◽  
Detection System ◽  
Transgenic Fish ◽  
Sybr Green ◽  
Wild Type ◽  
Quantitative Real Time Pcr ◽  
Oncorhynchus Masou ◽  
Cycle Threshold

<p>Identification of zygosity in transgenik fish is normally achieved by PCR analysis with genomic DNA template extracted from the tissue of progenies which are derived by mating the transgenic fish and wild-type counterpart.  This method needs relatively large amounts of fish material and is time- and labor-intensive. New approaches addressing this problem could be of great help for fish biotechnologists.  In this experiment, we applied a quantitative real-time PCR (qr-PCR) method to analyze zygosity in a stable line of transgenic zebrafish (<em>Danio rerio</em>) carrying masu salmon, <em>Oncorhynchus masou</em> D6-desaturase-like gene. The qr-PCR was performed using iQ SYBR Green Supermix in the iCycler iQ Real-time PCR Detection System (Bio-Rad Laboratories, USA).  Data were analyzed using the comparative cycle threshold method.  The results demonstrated a clear-cut identification of all transgenic fish (<em>n</em>=20) classified as a homozygous or heterozygous.  Mating of those fish with wild-type had revealed transgene transmission to the offspring following expected Mendelian laws. Thus, we found that the qTR-PCR to be effective for a rapid and precise determination of zygosity in transgenic fish. This technique could be useful in the establishment of breeding programs for mass transgenic fish production and in experiments in which zygosity effect could have a functional impact.</p> <p>Keywords: quantitative real-time PCR; zygosity; transgenic fish; mass production</p> <p> </p> <p>ABSTRAK</p> <p>Identifikasi sigositas ikan transgenik biasanya dilakukan menggunakan analisa PCR dengan cetakan DNA genomik yang diekstraksi dari jaringan ikan hasil persilangan antara ikan transgenik dan ikan normal.   Metode ini memerlukan ikan dalam jumlah yang banyak, dan juga waktu serta tenaga.  Pendekatan baru untuk mengatasi masalah tersebut akan memberikan manfaat besar kepada peneliti bioteknologi perikanan.  Pada penelitian ini, kami menggunakan metode PCR real-time kuantitatif (krt-PCR) untuk menganalisa sigositas pada satu strain ikan zebra (<em>Danio rerio</em>)<em> </em>transgenik yang membawa gen D6-desaturase-like dari ikan salmon masu, <em>Oncorhynchus masou</em>.<em>  </em>krt-PCR dilakukan menggunakan <em>iQ SYBR Green Supermix</em> pada mesin <em>iCycler iQ Real-time PCR Detection system</em> (Bio-Rad Laboratories, USA).  Data dianalisis menggunakan metode pembandingan nilai <em>cycle threshold</em>.  Hasil penelitian menunjukkan bahwa semua ikan transgenik (<em>n</em>=20) yang diidentifikasi dapat diklasifikasikan secara jelas sebagai ikan homosigot atau heterosigot.  Persilangan antara ikan transgenik tersebut dengan ikan normal menunjukkan transmisi transgen ke keturunannya mengikuti hukum segregasi Mendel.  Dengan demikian, metode krt-PCR adalah efektif untuk penentuan sigositas secara cepat dan tepat pada ikan transgenik.  Teknik ini dapat berguna dalam program produksi ikan transgenik secara massal dan dalam percobaan dimana faktor sigositas memberikan pengaruh nyata.</p> Kata kunci: kuantitatif real-time PCR; sigositas, ikan transgenik; produksi massal

scholarly journals Lead and cadmium influences on the metallothionein expression level in Lymnaea stagnalis L. adults

1970 ◽  
pp. 281-285
Author(s):  
S. E. Dromashko ◽  
S. N. Shevtsova ◽  
A. S. Babenko
Keyword(s):  
Gene Expression ◽  
Real Time ◽  
Real Time Pcr ◽  
Lymnaea Stagnalis ◽  
Test System ◽  
Expression Level ◽  
Pond Snail ◽  
Cadmium Ions ◽  
Lead And Cadmium ◽  
Expression Evaluation

Aim. The article deals with the assessment of some heavy metals effects on metallothionein expression in such a test system as the great pond snail L. stagnalis. Methods. There are methods of mollusk specimen cultivation and gene expression level estimation (PCR in real-time mode), as well as data analysis. Results. A significant increase in MT gene expression was observed in both young (4.5 months) and older (7 months) mollusks on the 7th day of exposure to 0.003 mg/L of lead ions as well as significant increase in MT gene expression was observed in older mollusks on the 7th day of exposure to 0.0001–0.001 mg/L of cadmium ions. Conclusions. The expediency of L. stagnalis adults using at the age of 3 to 5 months with other test systems for biotesting liquid metal-containing waste based on the gene expression evaluation in a short (no more than 7 day) experiment using the Real Time PCR has been shown.Keywords: Lymnaea stagnalis, lead/cadmium, metallothioneins, gene expression, real time PCR.

scholarly journals Selection and validation of reference genes for quantitative Real-Time PCR in Arabis alpina

2019 ◽  
Author(s):  
Lisa Stephan ◽  
Vicky Tilmes ◽  
Martin Hülskamp
Keyword(s):  
Gibberellic Acid ◽  
Real Time ◽  
Real Time Pcr ◽  
Reference Genes ◽  
Model Organism ◽  
Quantitative Real Time Pcr ◽  
Qpcr Data ◽  
Wide Range ◽  
Arabis Alpina ◽  
Acid Treatments

AbstractArabis alpina is a perennial arctic-alpine plant and an upcoming model organism for genetics and molecular biology for the Brassicaceae family. One essential method for most molecular approaches is the analysis of gene expression by quantitative Real-Time PCR (qPCR). For the normalisation of expression data in qPCR experiments, it is essential to use reliable reference genes that are not affected under a wide range of conditions. In this study we establish a set of 15 A. alpina reference genes that were tested under different conditions including cold, drought, heat, salt and gibberellic acid treatments. Data analyses with geNORM, BestKeeper and NormFinder revealed the most stable reference genes for the tested conditions: RAN3, HCF and PSB33 are most suitable for cold treatments; UBQ10 and TUA5 for drought; RAN3, PSB33 and EIF4a for heat; CAC, TUA5, ACTIN 2 and PSB33 for salt and PSB33 and TUA5 for gibberellic acid treatments. CAC and ACTIN 2 showed the least variation over all tested samples. In addition, we show that two reference genes are sufficient to normalize qPCR data under our treatment conditions. In future studies, these reference genes can be used for an adequate normalisation and thus help to generate high quality qPCR data in A. alpina.

scholarly journals KLK11 promotes the activation of mTOR and protein synthesis to facilitate cardiac hypertrophy

2021 ◽  
Vol 21 (1) ◽  
Author(s):  
Yi Wang ◽  
Hongjuan Liao ◽  
Yueheng Wang ◽  
Jinlin Zhou ◽  
Feng Wang ◽  
...  
Keyword(s):  
Protein Synthesis ◽  
Angiotensin Ii ◽  
Cardiac Hypertrophy ◽  
Western Blot ◽  
Real Time ◽  
Real Time Pcr ◽  
Mtor Signaling ◽  
Heart Weight ◽  
Cardiomyocyte Hypertrophy ◽  
Quantitative Real Time Pcr

Abstract Background Cardiovascular diseases have become the leading cause of death worldwide, and cardiac hypertrophy is the core mechanism underlying cardiac defect and heart failure. However, the underlying mechanisms of cardiac hypertrophy are not fully understood. Here we investigated the roles of Kallikrein 11 (KLK11) in cardiac hypertrophy. Methods Human and mouse hypertrophic heart tissues were used to determine the expression of KLK11 with quantitative real-time PCR and western blot. Mouse cardiac hypertrophy was induced by transverse aortic constriction (TAC), and cardiomyocyte hypertrophy was induced by angiotensin II. Cardiac function was analyzed by echocardiography. The signaling pathway was analyzed by western blot. Protein synthesis was monitored by the incorporation of [3H]-leucine. Gene expression was analyzed by quantitative real-time PCR. Results The mRNA and protein levels of KLK11 were upregulated in human hypertrophic hearts. We also induced cardiac hypertrophy in mice and observed the upregulation of KLK11 in hypertrophic hearts. Our in vitro experiments demonstrated that KLK11 overexpression promoted whereas KLK11 knockdown repressed cardiomyocytes hypertrophy induced by angiotensin II, as evidenced by cardiomyocyte size and the expression of hypertrophy-related fetal genes. Besides, we knocked down KLK11 expression in mouse hearts with adeno-associated virus 9. Knockdown of KLK11 in mouse hearts inhibited TAC-induced decline in fraction shortening and ejection fraction, reduced the increase in heart weight, cardiomyocyte size, and expression of hypertrophic fetal genes. We also observed that KLK11 promoted protein synthesis, the key feature of cardiomyocyte hypertrophy, by regulating the pivotal machines S6K1 and 4EBP1. Mechanism study demonstrated that KLK11 promoted the activation of AKT-mTOR signaling to promote S6K1 and 4EBP1 pathway and protein synthesis. Repression of mTOR with rapamycin blocked the effects of KLK11 on S6K1 and 4EBP1 as well as protein synthesis. Besides, rapamycin treatment blocked the roles of KLK11 in the regulation of cardiomyocyte hypertrophy. Conclusions Our findings demonstrated that KLK11 promoted cardiomyocyte hypertrophy by activating AKT-mTOR signaling to promote protein synthesis.

Quantitative real-time PCR assay to detect transgene copy number in cotton (Gossypium hirsutum)

2008 ◽  
Vol 375 (1) ◽  
pp. 150-152 ◽  
Author(s):  
Cheng Xin Yi ◽  
Jun Zhang ◽  
Ka Man Chan ◽  
Xiao Kun Liu ◽  
Yan Hong
Keyword(s):  
Gossypium Hirsutum ◽  
Real Time ◽  
Real Time Pcr ◽  
Copy Number ◽  
Quantitative Real Time Pcr ◽  
Pcr Assay ◽  
Transgene Copy Number
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