Comparison of the efficiency and relevancy of reference genes in crayfish tissue samples / Kerevit doku örneklerinde referans genlerin geçerlilik ve etkinliğinin karşılaştırılması

2016 ◽  
Vol 41 (3) ◽  
Author(s):  
Bora Ergin ◽  
Nuhan Purali
Keyword(s):  
Reference Gene ◽  
Ribosomal Rna ◽  
Heart Muscle ◽  
Reference Genes ◽  
Binding Protein ◽  
Expression Level ◽  
Tissue Samples ◽  
18S Ribosomal Rna ◽  
Melting Curves ◽  
Peptidylprolyl Isomerase

AbstractObjective: Relevancy of the reference genes have not been investigated in crayfish tissue samples. Various genes have been proposed for establishing a reference level to analyze qPCR results obtained in different tissue samples. However, majority of those genes have been investigated solely in mammalian species. There is no report related to the relevancy and the efficiency of those common reference genes in crayfish tissue samples yet. The primary aim of the present work is to compare the expression level of a set of reference genes in various crayfish tissue samples to establish a reliable reference gene for quantitative expression level comparisons.Methods: cDNA copies were synthesized by RT-PCR from equalised total RNA samples of nine different tissue samples. qPCR were performed on cDNA samples by using four different primer pairs specific for β-actin, Peptidylprolyl Isomerase B, Sarcoplasmic Calcium Binding Protein and 18S ribosomal RNA genes.Results: qActin2 amplified gene fragments for β-actin in all samples. Cycle time of the amplification reaction differed substantially between the investigated tissue types. A similar difference was observed between the melting curves of the products. The primer pair PPIB amplified gene fragments for Peptidylprolyl Isomerase B only in heart, muscle and intestine samples but not in the rest of the tissue samples. Further, both amplification and melting curves for heart, muscle and intestine were different. qSCP amplified some gene fragments for Sarcoplasmic Reticulum Binding Protein only in muscle, heart and ganglion samples. The 18S_1 amplified fragments for 18S. Amplification curves the samples superimposed on each other. Melting curves of the products were very similar. Variance of the obtained data was the lowest for 18S ribosomal RNA.Conclusion: In refer to present results; it is conceivable to propose that among all the genes investigated in the present study, expression level of ribosomal 18S RNA is the most homogeneously distributed one within the body parts of the crayfish. Further, it is minimally affected by extreme biological changes such as molting. Thus, 18S Ribosomal RNA would be suggested as a reference gene to be used when investigating tissue specific expression level of a gene in the crayfish.

scholarly journals 18S Ribosomal RNA Evaluation as Preanalytical Quality Control for Animal DNA

2016 ◽  
Vol 2016 ◽  
pp. 1-6
Author(s):  
Cory Ann Leonard ◽  
Marina L. Meli ◽  
Marilisa Novacco ◽  
Nicole Borel
Keyword(s):  
Ribosomal Rna ◽  
18S Rrna ◽  
Extraction Methods ◽  
Spectrophotometric Analysis ◽  
Rrna Gene ◽  
Host Animal ◽  
Tissue Samples ◽  
18S Ribosomal Rna ◽  
Qpcr Analysis ◽  
Ffpe Samples

The 18S ribosomal RNA (rRNA) gene is present in all eukaryotic cells. In this study, we evaluated the use of this gene to verify the presence of PCR-amplifiable host (animal) DNA as an indicator of sufficient sample quality for quantitative real-time PCR (qPCR) analysis. We compared (i) samples from various animal species, tissues, and sample types, including swabs; (ii) multiple DNA extraction methods; and (iii) both fresh and formalin-fixed paraffin-embedded (FFPE) samples. Results showed that 18S ribosomal RNA gene amplification was possible from all tissue samples evaluated, including avian, reptile, and FFPE samples and most swab samples. A single swine rectal swab, which showed sufficient DNA quantity and the demonstrated lack of PCR inhibitors, nonetheless was negative by 18S qPCR. Such a sample specifically illustrates the improvement of determination of sample integrity afforded by inclusion of 18S rRNA gene qPCR analysis in addition to spectrophotometric analysis and the use of internal controls for PCR inhibition. Other possible applications for the described 18S rRNA qPCR are preselection of optimal tissue specimens for studies or preliminary screening of archived samples prior to acceptance for biobanking projects.

scholarly journals Selection of Suitable Reference Genes for qPCR Gene Expression Analysis of HepG2 and L02 in Four Different Liver Cell Injured Models

2020 ◽  
Vol 2020 ◽  
pp. 1-11
Author(s):  
Jiyu Chen ◽  
Zhenzhen Bao ◽  
Yanli Huang ◽  
Zhenglong Wang ◽  
Yucheng Zhao
Keyword(s):  
Reference Gene ◽  
Reference Genes ◽  
Cell Types ◽  
Expression Level ◽  
Suitable Reference Gene ◽  
Drug Treatments ◽  
Tata Box Binding Protein ◽  
The Stability ◽  
Beta 2 Microglobulin ◽  
Suitable Reference

Quantitative real-time PCR (qPCR) has become a widely used approach to analyze the expression level of selected genes. However, owing to variations in cell types and drug treatments, a suitable reference gene should be selected according to special experimental design. In this study, we investigated the expression level of ten candidate reference genes in hepatoma carcinoma cell (HepG2) and human hepatocyte cell line (L02) treated with ethanol (EtOH), hydrogen peroxide (H2O2), acetaminophen (APAP), and carbon tetrachloride (CCl4), respectively. To analyze raw cycle threshold values (Cp values) from qPCR run, three reference gene validation programs, including Bestkeeper, geNorm, and NormFinder, were used to evaluate the stability of ten candidate reference genes. The results showed that TATA-box binding protein (TBP) and tubulin beta 2a (TUBB2a) presented the highest stability for normalization under different treatments and were regarded as the most suitable reference genes of HepG2 and L02. In addition, this study not only identified the most stable reference genes of each treatment, but also suggested that β-actin (ACTB), glyceraldehade-3-phosphate dehydrogenase (GAPDH), tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein zeta (YWHAZ), and beta-2 microglobulin (B2M) were the least stable reference genes in HepG2 and L02. This work was the first report to systematically explore the stability of reference genes in injured models of HepG2 and L02.

Standardization of Reference Genes in Silkworm, Bombyx Mori

2011 ◽  
Vol 175-176 ◽  
pp. 67-71 ◽  
Author(s):  
Ran Peng ◽  
Bao Jin Su ◽  
Guo Dong Zhao ◽  
Xing Ji ◽  
Si Si Zhao ◽  
...  
Keyword(s):  
Bombyx Mori ◽  
Reference Gene ◽  
Reference Genes ◽  
Target Gene ◽  
Model Organism ◽  
Production Quality ◽  
Research Field ◽  
Expression Level ◽  
Mrna Transcription ◽  
Study Gene Expression

The Bombyx mori serves as model organism among the Lepidoptera insects. In the post-genomic era, in order to study gene function, the profiling of mRNA transcription has become a popular research field. Real-time quantitative RT-PCR (qRT-PCR) has become established as the sensitive method for detecting the expression level of low abundance mRNA, and it usually chooses one or several reference genes to standardize the expression level of target gene. Since the changes in amplification of reference gene can reflect the changes of RNA production, quality or cDNA synthesis efficiency. So choosing an appropriate reference gene can reduce the differences between tested samples. Based on the comparison of Standardization of three frequently-used reference genes (GAPDH, Actin-3, 28srRNA), and decide which is the best way to study gene expression level in silkworm, Bombyx mori.

scholarly journals Selection of reference genes for quantitative real-time PCR analysis in halophytic plantRhizophora apiculata

PeerJ ◽  
2018 ◽  
Vol 6 ◽  
pp. e5226 ◽  
Author(s):  
Ankush Ashok Saddhe ◽  
Manali Ramakant Malvankar ◽  
Kundan Kumar
Keyword(s):  
Gene Expression ◽  
Salt Stress ◽  
Real Time ◽  
Reference Gene ◽  
Reference Genes ◽  
Candidate Reference Gene ◽  
Expression Level ◽  
Bisphosphate Carboxylase ◽  
Qrt Pcr ◽  
Selection Of

Rhizophora apiculatais a halophytic, small mangrove tree distributed along the coastal regions of the tropical and subtropical areas of the world. They are natural genetic reservoirs of salt adaptation genes and offer a unique system to explore adaptive mechanisms under salinity stress. However, there are no reliable studies available on selection and validation of reference genes for quantitative real-time polymerase chain reaction (qRT-PCR) inR. apiculataphysiological tissues and in salt stress conditions. The selection of appropriate candidate reference gene for normalization of qRT-PCR data is a crucial step towards relative analysis of gene expression. In the current study, seven genes such as elongation factor 1α (EF1α), Ubiquitin (UBQ), β-tubulin (β-TUB), Actin (ACT), Ribulose1,5-bisphosphate carboxylase/oxygenase (rbcL), Glyceraldehyde 3-phosphate dehydrogenase (GAPDH), and 18S rRNA (18S) were selected and analyzed for their expression stability. Physiological tissues such as leaf, root, stem, and flower along with salt stress leaf samples were used for selection of candidate reference genes. The high-quality expression data was obtained from biological replicates and further analyzed using five different programs such as geNorm, NormFinder, BestKeeper, Delta Ct and RefFinder. All algorithms comprehensively rankedEF1α followed byACTas the most stable candidate reference genes inR. apiculataphysiological tissues. Moreover, β-TUBand 18S were ranked as moderately stable candidate reference genes, while GAPDH andrbcLwere least stable reference genes. Under salt stress,EF1α was comprehensively recommended top-ranked candidate reference gene followed byACTand 18S. In order to validate the identified most stable candidate reference genes,EF1α,ACT, 18S andUBQwere used for relative gene expression level of sodium/proton antiporter (NHX) gene under salt stress. The expression level ofNHXvaried according to the internal control which showed the importance of selection of appropriate reference gene. Taken together, this is the first ever systematic attempt of selection and validation of reference gene for qRT-PCR inR. apiculataphysiological tissues and in salt stress. This study would promote gene expression profiling of salt stress tolerance related genes inR. apiculata.

scholarly journals Molecular Identification of Trypanosoma evansi Isolated from Arabian Camels (Camelus dromedarius) in Riyadh and Al-Qassim, Saudi Arabia

Animals ◽  
2021 ◽  
Vol 11 (4) ◽  
pp. 1149
Author(s):  
Dina M. Metwally ◽  
Isra M. Al-Turaiki ◽  
Najwa Altwaijry ◽  
Samia Q. Alghamdi ◽  
Abdullah D. Alanazi
Keyword(s):  
Saudi Arabia ◽  
Infection Rate ◽  
Ribosomal Rna ◽  
Phylogenetic Analyses ◽  
Trypanosoma Evansi ◽  
Camelus Dromedarius ◽  
18S Ribosomal Rna ◽  
18S Ribosomal Rna Gene ◽  
Polymerase Chain ◽  
Pcr Targeting

We analyzed the blood from 400 one-humped camels, Camelus dromedarius (C. dromedarius), in Riyadh and Al-Qassim, Saudi Arabia to determine if they were infected with the parasite Trypanosoma spp. Polymerase chain reaction (PCR) targeting the internal transcribed spacer 1 (ITS1) gene was used to detect the prevalence of Trypanosoma spp. in the camels. Trypanosoma evansi (T. evansi) was detected in 79 of 200 camels in Riyadh, an infection rate of 39.5%, and in 92 of 200 camels in Al-Qassim, an infection rate of 46%. Sequence and phylogenetic analyses revealed that the isolated T. evansi was closely related to the T. evansi that was detected in C. dromedarius in Egypt and the T. evansi strain B15.1 18S ribosomal RNA gene identified from buffalo in Thailand. A BLAST search revealed that the sequences are also similar to those of T. evansi from beef cattle in Thailand and to T. brucei B8/18 18S ribosomal RNA from pigs in Nigeria.

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