Selection and validation of reference genes in all-red Amaranth (Amaranthus tricolor L.) seedlings under different culture conditions

2021 ◽  
pp. 1-10
Author(s):  
Fang Xiao ◽  
Youfeng Zheng ◽  
Jialan Chen ◽  
Chunli Zhao ◽  
He Chen ◽  
...  
Keyword(s):  
Reference Genes ◽  
Culture Conditions ◽  
Amaranthus Tricolor

scholarly journals Selection and validation of reference genes for quantitative real-time PCR in the green microalgae Tetraselmis chui

PLoS ONE ◽  
2021 ◽  
Vol 16 (1) ◽  
pp. e0245495
Author(s):  
Sonia Torres ◽  
Carmen Lama ◽  
Lalia Mantecón ◽  
Emmanouil Flemetakis ◽  
Carlos Infante
Keyword(s):  
Real Time ◽  
Reference Genes ◽  
Large Scale ◽  
Critical Factor ◽  
Continuous Phase ◽  
Culture Conditions ◽  
Green Microalgae ◽  
Industrial Facilities ◽  
Reverse Transcription Pcr ◽  
Tetraselmis Chui

Quantitative real-time reverse transcription PCR (RT-qPCR) is a highly sensitive technique that can be applied to analyze how genes are modulated by culture conditions, but identification of appropriate reference genes for normalization is a critical factor to be considered. For this reason, the expression stability of 18 candidate reference genes was evaluated for the green microalgae Tetraselmis chui using the widely employed algorithms geNorm, NormFinder, BestKeeper, the comparative ΔCT method, and RefFinder. Microalgae samples were collected from large scale outdoor photobioreactors during the growing phase (OUT_GP), and during the semi-continuous phase at different times of the day (OUT_DC). Samples from standard indoor cultures under highly controlled conditions (IND) were also collected to complement the other data. Different rankings for the candidate reference genes were obtained depending on the culture conditions and the algorithm employed. After comparison of the achieved ranks with the different methods, the references genes selected for samples from specific culture conditions were ALD and EFL in OUT_GP, RPL32 and UBCE in OUT_DC, and cdkA and UBCE in IND. Moreover, the genes EFL and cdkA or EFL and UBCE appeared as appropriate combinations for pools generated from all samples (ALL). Examination in the OUT_DC cultures of genes encoding the large and small subunits of ADP-glucose pyrophosphorylase (AGPL and AGPS, respectively) confirmed the reliability of the identified reference genes, RPL32 and UBCE. The present study represents a useful contribution for studies of gene expression in T. chui, and also represents the first step to set-up an RT-qPCR platform for quality control of T. chui biomass production in industrial facilities.

scholarly journals Chlamydia psittaci reference genes for normalisation of expression data differ depending on the culture conditions and selected time points during the chlamydial replication cycle

2016 ◽  
Vol 60 (4) ◽  
pp. 403-409
Author(s):  
Sarah Van Lent ◽  
Daisy Vanrompay
Keyword(s):  
Reference Genes ◽  
Normal Growth ◽  
Cell Interaction ◽  
Culture Conditions ◽  
Study Data ◽  
Chlamydia Psittaci ◽  
Economic Losses ◽  
Developmental Cycle ◽  
Time Points ◽  
Obligate Intracellular Pathogen

Abstract Introduction: Chlamydia psittaci is a gram-negative obligate intracellular pathogen of birds. Poultry infections lead to economic losses and can be transmitted to humans. No vaccine is available and the bacterium-host cell interaction is not completely understood. Replicating bacteria cause pneumonia, but C. psittaci can also be non-replicating and persistent inside the cytoplasm of avian cells. RT-qPCR provides insight into the molecular pathogenesis of both active replicating and persistent Chlamydia psittaci in birds, but requires identification of stably expressed reference genes to avoid biases. Material and Methods: We investigated the expression stability of 10 C. psittaci candidate reference genes for gene expression analysis during normal growth and penicillin-induced persistence. C. psittaci Cal10 was cultured in HeLa229 and RNA was extracted. The expression level of each candidate was examined by RT-qPCR and Cq values were analysed using geNorm. Results: The genes tyrS, gidA, radA, and 16S rRNA ranked among the most stably expressed. The final selected reference genes differed according to the bacterial growth status (normal growth versus persistent status), and the time points selected during the duration of the normal chlamydial developmental cycle. Conclusion: The study data show the importance of systematic validation of reference genes to confirm their stability within the strains and under the conditions selected.

scholarly journals New Reference Genes for qRT-PCR Analysis as a Potential Target for Identification of Trichophyton verrucosum in Different Culture Conditions

Pathogens ◽  
2021 ◽  
Vol 10 (11) ◽  
pp. 1361
Author(s):  
Sebastian Gnat ◽  
Dominik Łagowski ◽  
Aneta Nowakiewicz ◽  
Aleksandra Trościańczyk ◽  
Mariusz Dyląg
Keyword(s):  
Gene Expression ◽  
Reference Genes ◽  
Housekeeping Gene ◽  
Culture Conditions ◽  
Pcr Analysis ◽  
Trichophyton Verrucosum ◽  
Diagnostic Laboratory ◽  
Reliable Technique ◽  
Qrt Pcr ◽  
Diagnostic Difficulties

Dermatophytes are a group of filamentous fungi infecting skin, hair, and nails that raise great diagnostic difficulties. qRT-PCR is a reliable technique for quantifying gene expression with increasingly frequent use in mycological diagnostics. Knowledge of genes and molecular markers with potential to be used in the identification of dermatophytes is of great importance for the development of this branch of diagnostics. In this article, the suitability of six candidate reference genes (TUBB, ACTB, ADPRF, RPL2, SDHA, and EEF1A1) was investigated for gene expression analysis in the dermatophyte Trichophyton verrucosum, which was cultured in various mycological media that are commonly used in a diagnostic laboratory, i.e., Sabouraud, potato dextrose, and keratin-supplemented MM-Cove. The different culture conditions are extremely important factors for the growth and physiology of dermatophytes. Gene expression stability was evaluated using geNorm, NormFinder, BestKeeper, and RefFinder algorithms. Regarding the stability of expression, SDHA was the most stable housekeeping gene; hence, this gene is recommended for future qRT-PCR studies on T. verrucosum strains. These results allow us to conclude that the SDHA gene can be an additional good candidate as an identification target in the qRT-PCR technique.

scholarly journals Validation of Reference Genes for Transcriptional Analyses in Pleurotus ostreatus by Using Reverse Transcription-Quantitative PCR

2015 ◽  
Vol 81 (12) ◽  
pp. 4120-4129 ◽  
Author(s):  
Raúl Castanera ◽  
Leticia López-Varas ◽  
Antonio G. Pisabarro ◽  
Lucía Ramírez
Keyword(s):  
Quantitative Pcr ◽  
Reverse Transcription ◽  
Pleurotus Ostreatus ◽  
Reference Genes ◽  
Time Course ◽  
Extracellular Enzymes ◽  
Expression Patterns ◽  
Model Organism ◽  
Culture Conditions ◽  
Content Type

ABSTRACTRecently, the lignin-degrading basidiomycetePleurotus ostreatushas become a widely used model organism for fungal genomic and transcriptomic analyses. The increasing interest in this species has led to an increasing number of studies analyzing the transcriptional regulation of multigene families that encode extracellular enzymes. Reverse transcription (RT) followed by real-time PCR is the most suitable technique for analyzing the expression of gene sets under multiple culture conditions. In this work, we tested the suitability of 13 candidate genes for their use as reference genes inP. ostreatustime course cultures for enzyme production. We applied three different statistical algorithms and obtained a combination of stable reference genes for optimal normalization of RT-quantitative PCR assays. This reference index can be used for future transcriptomic analyses and validation of transcriptome sequencing or microarray data. Moreover, we analyzed the expression patterns of a laccase and a manganese peroxidase (lacc10andmnp3, respectively) in lignocellulose and glucose-based media using submerged, semisolid, and solid-state fermentation. By testing different normalization strategies, we demonstrate that the use of nonvalidated reference genes as internal controls leads to biased results and misinterpretations of the biological responses underlying expression changes.

A new set of reference genes for RT-qPCR assays in the yeast Dekkera bruxellensis

2012 ◽  
Vol 58 (12) ◽  
pp. 1362-1367 ◽  
Author(s):  
Will de Barros Pita ◽  
Fernanda Cristina Bezerra Leite ◽  
Anna Theresa de Souza Liberal ◽  
Luciana Filgueira Pereira ◽  
Marcelo Falsarella Carazzolle ◽  
...  
Keyword(s):  
Gene Expression ◽  
Reference Gene ◽  
Reference Genes ◽  
Gene Expression Analysis ◽  
Regulation Of Gene Expression ◽  
Culture Conditions ◽  
Mrna Levels ◽  
Dekkera Bruxellensis ◽  
Expression Of Genes ◽  
Normalization Factors

The yeast Dekkera bruxellensis has been recently regarded as an important microorganism for bioethanol production owing to its ability to convert glucose, sucrose, and cellobiose to ethanol. The aim of this work was to validate a new set of reference genes for gene expression analysis by quantitative real-time PCR in D. bruxellensis and compare the influence of the method of choice for quantification of mRNA levels with the reliability of our data. Three candidate reference genes, DbEFA1, DbEFB1, and DbYNA1, were used in a quantitative analysis of 4 genes of interest, DbYNR1, DbTPS1, DbADH7, and DbUBA4, based on an approach for calculating the normalization factors by means of the geNorm applet. Each reference gene was also individually used for a [Formula: see text] (comparative Cq method) calculation of the relative expression of genes of interest. Our results showed that the 3 reference genes provided enough stability and were complementary to the normalization factors method in different culture conditions. This work was able to confirm the usefulness of a previously reported reference gene, EFA1/TEF1, and increased the set of possible reference genes in D. bruxellensis to 4. Moreover, this can improve the reliability of the analysis of the regulation of gene expression in the industrial yeast D. bruxellensis.

scholarly journals Effect of Culture Conditions on Reference Genes Expression in Placenta-derived Stem Cells

2009 ◽  
Vol 2 (1) ◽  
pp. 69-75 ◽  
Author(s):  
Crescenzio F. Minervini ◽  
Masanori Izumi ◽  
Toshio Miki
Keyword(s):  
Stem Cells ◽  
Reference Genes ◽  
Culture Conditions ◽  
Genes Expression
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