scholarly journals Evaluation of the expression stability of reference genes in Apis mellifera under pyrethroid treatment

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Przemysław Wieczorek ◽  
Patryk Frąckowiak ◽  
Aleksandra Obrępalska-Stęplowska
Keyword(s):  
Apis Mellifera ◽  
Agricultural Production ◽  
Reference Genes ◽  
Housekeeping Genes ◽  
Chain Reaction ◽  
Experimental Conditions ◽  
Breeding Lines ◽  
Lambda Cyhalothrin ◽  
Polymerase Chain ◽  
External Stimuli

Abstract Honeybees (Apis mellifera L.), which unquestionably play an economically important role in pollination and agricultural production, are at risk of decline. To study changes in gene expression in insects upon exposure to pesticides or other external stimuli, appropriate reference genes are required for data normalization. Since there is no such gene that is absolutely invariable under all experimental conditions, the aim of this study was to identify the most stable targets suitable for subsequent normalization in quantitative experiments based on real-time polymerase chain reaction in honeybee research. Here, we evaluated the expression of fifteen candidate housekeeping genes from three breeding lines of honeybees treated with pyrethroids to identify the most stable genes. The tested insects were exposed to deltamethrin or lambda-cyhalothrin, and then, changes in the accumulation of selected transcripts were assessed, followed by statistical analyses. We concluded that AmRPL32, AmACT and AmRPL13a were the commonly recorded most stable genes in honeybees treated with the selected pyrethroids.

scholarly journals TATA box binding protein and ribosomal protein 4 are suitable reference genes for normalization during quantitative polymerase chain reaction study in bovine mesenchymal stem cells

2020 ◽  
Vol 33 (12) ◽  
pp. 2021-2030
Author(s):  
Si-Jung Jang ◽  
Ryoung-Hoon Jeon ◽  
Hwan-Deuk Kim ◽  
Jong-Chan Hwang ◽  
Hyeon-Jeong Lee ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Ribosomal Protein ◽  
Reference Genes ◽  
Binding Protein ◽  
Quantitative Polymerase Chain Reaction ◽  
Tata Box ◽  
Chain Reaction ◽  
Experimental Conditions ◽  
Polymerase Chain ◽  
Tata Box Binding Protein

Objective: Quantitative polymerase chain reaction (qPCR) has been extensively used in the field of mesenchymal stem cell (MSC) research to elucidate their characteristics and clinical potential by normalization of target genes against reference genes (RGs), which are believed to be stably expressed irrespective of various experimental conditions. However, the expression of RGs is also variable depending on the experimental conditions, which may lead to false or contradictory conclusions upon normalization. Due to the current lack of information for a clear list of stable RGs in bovine MSCs, we conducted this study to identify suitable RGs in bovine MSCs.Methods: The cycle threshold values of ten traditionally used RGs (18S ribosomal RNA [18S], beta-2-microglobulin [B2M], H2A histone family, member Z [H2A], peptidylprolyl isomerase A [PPIA], ribosomal protein 4 [RPL4], succinate dehydrogenase complex, subunit A [SDHA], beta actin [ACTB], glyceraldehyde-3-phosphate dehydrogenase [GAPDH], TATA box binding protein [TBP], and hypoxanthine phosphoribosyltrasnfrase1 [HPRT1]) in bovine bone marrow-derived MSCs (bBMMSCs) were validated for their stabilities using three types of RG evaluation algorithms (geNorm, Normfinder, and Bestkeeper). The effect of validated RGs was then verified by normalization of lineage-specific genes (fatty acid binding protein 4 [FABP4] and osteonectin [ON]) expressions during differentiations of bBMMSCs or POU class 5 homeobox 1 (OCT4) expression between bBMMSCs and dermal skins.Results: Based on the results obtained for the three most stable RGs from geNorm (TBP, RPL4, and H2A), Normfinder (TBP, RPL4, and SDHA), and Bestkeeper (TBP, RPL4, and SDHA), it was comprehensively determined that TBP and RPL4 were the most stable RGs in bBMMSCs. However, traditional RGs were suggested to be the least stable (18S) or moderately stable (GAPDH and ACTB) in bBMMSCs. Normalization of FABP4 or ON against TBP, RPL4, and 18S presented significant differences during differentiation of bBMMSCs. However, although significantly low expression of OCT4 was detected in dermal skins compared to that in bBMMSCs when TBP and RPL4 were used in normalization, normalization against 18S exhibited no significance.Conclusion: This study proposes that TBP and RPL4 were suitable as stable RGs for qPCR study in bovine MSCs.

scholarly journals Reference genes for livestock gene expression profiling – Literature review

2017 ◽  
pp. 81-89
Author(s):  
Ádám Simon ◽  
Zsuzsa Zakarné Aszalós ◽  
András Jávor ◽  
Levente Czeglédi
Keyword(s):  
Gene Expression ◽  
Polymerase Chain Reaction ◽  
Literature Review ◽  
Real Time ◽  
Reference Genes ◽  
Chain Reaction ◽  
Experimental Conditions ◽  
Genes Encoding ◽  
Polymerase Chain ◽  
Protein Components

Quantitative real-time polymerase chain reaction (qPCR) is an essential tool for understanding animal cell’s response to developmental progression or to different experimental conditions at gene expression level. However the reliability of this method heavily lies on proper normalization (measuring a target and a reference gene’s expression from the same sample to correct for technical related variations).Our literature review aimed to summarize the articles addressing the most important livestock species in regards of reference gene stability used as normalizers for quantitative real-time polymerase chain reaction experiments. Stably expressing reference genes were categorized into 14 distinct groups according to gene function. The number of reference genes tested and the publication numbers according to years and the ranking algorithms were also noted.Counting showed that genes encoding ribosomal protein components are ranked as most stable in majority of cases and therefore should be taking into account for qPCR stable normalizer gene finding experiments.

Validation of Reference Genes for Real-Time Polymerase Chain Reaction Studies in Atlantic Salmon

2006 ◽  
Vol 8 (4) ◽  
pp. 398-408 ◽  
Author(s):  
Sven Martin Jorgensen ◽  
Ellen Johanne Kleveland ◽  
Unni Grimholt ◽  
Tor Gjoen
Keyword(s):  
Polymerase Chain Reaction ◽  
Atlantic Salmon ◽  
Real Time ◽  
Reference Genes ◽  
Chain Reaction ◽  
Polymerase Chain

scholarly journals Investigation of Different Commercially Available Real Time-Polymerase Chain Reaction Kits for SARS-CoV-2 Diagnosis

2021 ◽  
Author(s):  
Rajeev Kumar Jain ◽  
Nagaraj Perumal ◽  
Rakesh Shrivastava ◽  
Kamlesh Kumar Ahirwar ◽  
Jaya Lalwani ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Real Time ◽  
Internal Control ◽  
Housekeeping Genes ◽  
Housekeeping Gene ◽  
Specific Gene ◽  
Rt Pcr ◽  
Chain Reaction ◽  
Gene Target ◽  
Polymerase Chain

Introduction: The whole world is facing an ongoing global health emergency of COVID-19 disease caused by the Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2). Real-Time Reverse Transcription-Polymerase Chain Reaction (RT-PCR) is a gold standard in the detection of SARS-CoV-2 infection. Presently, many single tube multiple gene target RT-PCR kits have been developed and are commercially available for Coronavirus Disease 2019 (COVID-19) diagnosis. Aim: To evaluate the performance of seven COVID-19 RT-PCR kits (DiagSure, Meril, VIRALDTECT II, TruPCR, Q-line, Allplex and TaqPath) which are commercially available for COVID-19 RT-PCR diagnosis. Materials and Methods: This observational study was conductedat the State Virology Laboratory (SVL), Gandhi Medical College, Bhopal, Madhya Pradesh, India. Seven commercially available kits have been evaluated on the basis of: (i) number of SARS-CoV-2 specific gene target; (ii) human housekeeping genes as internal control; (iii) RT-PCR run time; and (iv) kit performances to correctly detect SARS-CoV-2 positive and negative RNA samples. A total of 50 RNA samples (left over RNA) were included, master mix preparation, template addition and RT-PCR test has been performed according to kits literature. At the end of PCR run, mean and standard deviation of obtained cut-off of all kits were calculated using Microsoft Excel. Results: All seven RT-PCR kits performed satisfactory regarding the reproducibility and they could correctly identify 30 positive and 20 negative RNA samples. RNA samples (group C) having low viral loads with a high Cycle threshold (Ct) value (>30) were also detected by all these seven kits. Obtained Ct values of each group was in parallel range in comparison with the initial testing Ct values. Kits were found to be superior which contains primers and probes for three SARS-CoV-2 specific gene targets, have human housekeeping gene as internal control and taking less time to complete RT-PCR. Conclusion: All seven COVID-19 RT-PCR kits included in this study demonstrated satisfactory performance and can be used for the routine molecular diagnosis of COVID-19 disease.

Equivalence test in quantitative reverse transcription polymerase chain reaction: confirmation of reference genes suitable for normalization

2004 ◽  
Vol 335 (1) ◽  
pp. 1-9 ◽  
Author(s):  
Florian Haller ◽  
Bettina Kulle ◽  
Stefanie Schwager ◽  
Bastian Gunawan ◽  
Anja von Heydebreck ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Reverse Transcription ◽  
Reference Genes ◽  
Chain Reaction ◽  
Equivalence Test ◽  
Polymerase Chain

scholarly journals PRIMEval: Optimization and screening of multiplex oligonucleotide assays

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Rick Conzemius ◽  
Michaela Hendling ◽  
Stephan Pabinger ◽  
Ivan Barišić
Keyword(s):  
In Silico ◽  
Multiplex Polymerase Chain Reaction ◽  
Data Sets ◽  
Use Case ◽  
Chain Reaction ◽  
Experimental Conditions ◽  
Oligonucleotide Hybridization ◽  
Polymerase Chain ◽  
Derived Data ◽  
By Products

AbstractThe development of multiplex polymerase chain reaction and microarray assays is challenging due to primer dimer formation, unspecific hybridization events, the generation of unspecific by-products, primer depletion, and thus lower amplification efficiencies. We have developed a software workflow with three underlying algorithms that differ in their use case and specificity, allowing the complete in silico evaluation of such assays on user-derived data sets. We experimentally evaluated the method for the prediction of oligonucleotide hybridization events including resulting products and probes, self-dimers, cross-dimers and hairpins at different experimental conditions. The developed method allows explaining the observed artefacts through in silico WGS data and thermodynamic predictions. PRIMEval is available publicly at https://primeval.ait.ac.at.

Validation of reference genes for quantitative real‐time polymerase chain reaction in Drosophila melanogaster exposed to two chemicals

2019 ◽  
Vol 49 (6) ◽  
pp. 277-283 ◽  
Author(s):  
YeongHo Kim ◽  
YiSeul Kim ◽  
Donghun Kim ◽  
SeYeon Kim ◽  
GyeongJin Seo ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Drosophila Melanogaster ◽  
Real Time ◽  
Reference Genes ◽  
Chain Reaction ◽  
Polymerase Chain

scholarly journals Inter- and Intra-Species Diversity of Lactic Acid Bacteria in Apis mellifera ligustica Colonies

2020 ◽  
Vol 8 (10) ◽  
pp. 1578 ◽  
Author(s):  
Massimo Iorizzo ◽  
Gianfranco Pannella ◽  
Silvia Jane Lombardi ◽  
Sonia Ganassi ◽  
Bruno Testa ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Lactic Acid ◽  
Apis Mellifera ◽  
Lactic Acid Bacteria ◽  
Honey Bees ◽  
Rrna Gene ◽  
Chain Reaction ◽  
Rapd Pcr ◽  
The Social ◽  
Polymerase Chain

Lactic acid bacteria could positively affect the health of honey bees, including nutritional supplementation, immune system development and pathogen colonization resistance. Based on these considerations the present study evaluated predominant Lactic Acid Bacteria (LAB) species from beebread as well as from the social stomach and midgut of Apis mellifera ligustica honey bee foragers. In detail, for each compartment, the diversity in species and biotypes was ascertained through multiple culture-dependent approaches, consisting of Polymerase Chain Reaction-Denaturing Gradient Gel Electrophoresis (PCR-DGGE), 16S rRNA gene sequencing and Randomly Amplified Polymorphic DNA-Polymerase Chain Reaction (RAPD-PCR). The study of a lactic acid bacteria community, performed with PCR-DGGE and sequence analysis targeting the V1–V3 region of the 16S rRNA gene (rDNA), highlighted the presence of a few species, including Apilactobacillus kunkeei, Lactiplantibacillus plantarum, Fructobacillus fructosus, Levilactobacillus brevis and Lactobacillus delbrueckii subsp. lactis. Depending on the different compartments, diverse levels of biodiversity in species were found. Particularly, a very low inter-species biodiversity was detected in the midgut that was prevalently dominated by the presence of Apilactobacillus kunkeei. On the other hand, the beebread was characterized by a reasonable biodiversity showing the presence of five species and the predominance of Apilactobacillus kunkeei, Lactiplantibacillus plantarum and Fructobacillus fructosus. The RAPD-PCR analysis performed on the three predominant species allowed the differentiation into several biotypes for each species. Moreover, a relationship between biotypes and compartments has been detected and each biotype was able to express a specific biochemical profile. The biotypes that populated the social stomach and midgut were able to metabolize sugars considered toxic for bees while those isolated from beebread could contribute to release useful compounds with functional properties. Based on this knowledge, new biotechnological approaches could be developed to improve the health of honey bees and the quality of bee products.

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