Gene expression stability of candidate reference genes under different culture conditions for quantitative PCR in the raphidophyte Chattonella marina

Phycologia ◽  
2020 ◽  
Vol 59 (6) ◽  
pp. 556-565
Author(s):  
Koki Mukai ◽  
Yohei Shimasaki ◽  
Xuchun Qiu ◽  
Yoko Kato-Unoki ◽  
Kun Chen ◽  
...  
Keyword(s):  
Gene Expression ◽  
Quantitative Pcr ◽  
Reference Genes ◽  
Culture Conditions ◽  
Expression Stability ◽  
Chattonella Marina ◽  
Gene Expression Stability

scholarly journals Genome-Wide Identification of Reference Genes for Reverse-Transcription Quantitative PCR in Goat Rumen

Animals ◽  
2021 ◽  
Vol 11 (11) ◽  
pp. 3137
Author(s):  
Juan Zhao ◽  
Cheng Wang ◽  
Lin Zhang ◽  
Aiai Lei ◽  
Linjie Wang ◽  
...  
Keyword(s):  
Gene Expression ◽  
Quantitative Pcr ◽  
Reference Genes ◽  
Target Genes ◽  
Expression Profiles ◽  
Gene Expression Profiles ◽  
Expression Stability ◽  
Transport Pathway ◽  
Metabolic Role ◽  
Suitable Reference

As the largest chamber of the ruminant stomach, the rumen not only serves as the principal absorptive surface and nutrient transport pathway from the lumen into the animal, but also plays an important short-chain fatty acid (SCFA) metabolic role in addition to protective functions. Accurate characterization of the gene expression profiles of genes of interest is essential to the exploration of the intrinsic regulatory mechanisms of rumen development in goats. Thus, the selection of suitable reference genes (RGs) is an important prerequisite for real-time quantitative PCR (RT-qPCR). In the present study, 16 candidate RGs were identified from our previous transcriptome sequencing of caprine rumen tissues. The quantitative expressions of the candidate RGs were measured using the RT-qPCR method, and the expression stability of the RGs was assessed using the geNorm, NormFinder, and BestKeeper programs. GeNorm analysis showed that the M values were less than 0.5 for all the RGs except GAPT4, indicating that they were stably expressed in the rumen tissues throughout development. RPS4X and RPS6 were the two most stable RGs. Furthermore, the expressions of two randomly selected target genes (IGF1 and TOP2A), normalized by the selected most stable RGs (RPS4X and RPS6), were consistent with the results of RNA sequencing, while the use of GAPDH and ACTB as RGs resulted in altered profiles. Overall, RPS4X and RPS6 showed the highest expression stability and the lowest coefficients of variation, and could be used as the optimal reference combination for quantifying gene expression in rumen tissues via RT-qPCR analysis.

scholarly journals Selection and Validation of Appropriate Reference Genes for Gene Expression Studies in Schima Superba

2021 ◽  
Author(s):  
Zhongyi Yang ◽  
Rui Zhang ◽  
Zhichun Zhou
Keyword(s):  
Gene Expression ◽  
Reference Gene ◽  
Reference Genes ◽  
Geometric Mean ◽  
Gene Transcript ◽  
Expression Stability ◽  
Schima Superba ◽  
Expression Studies ◽  
Gene Expression Studies ◽  
Different Tissues

Abstract Background Quantitative real-time PCR (qRT-PCR) is a reliable and high-throughput technique for gene expression studies, but its accuracy depends on the expression stability of reference genes. Schima superba is a strong resistance and fast-growing timber specie. However, so far, reliable reference gene identifications have not been reported in S. superba. In this study, we screened and verified the stably expressed reference genes in different tissues of S. superba.Results Nineteen candidate reference genes were selected and evaluated for their expression stability in different tissues. Three software programs (geNorm, NormFinder, and BestKeeper) were used to evaluate the reference gene transcript stabilities, and comprehensive stability ranking was generated by the geometric mean method. Our results identified that SsuACT was the most stable reference gene, SsuACT + SsuRIB was the best reference genes combination for different tissues. Finally, the stable and less stable reference genes were verified using the SsuSND1 expression in different tissues.Conclusions This is the first report to verify the appropriate reference genes for normalizing gene expression in S. superba for different tissues, which will facilitate future elucidation of gene regulations in this species, and useful references for relative species.

scholarly journals Cloning and evaluation of reference genes for quantitative real-time PCR analysis inAmorphophallus

PeerJ ◽  
2017 ◽  
Vol 5 ◽  
pp. e3260 ◽  
Author(s):  
Kai Wang ◽  
Yi Niu ◽  
Qijun Wang ◽  
Haili Liu ◽  
Yi Jin ◽  
...  
Keyword(s):  
Gene Expression ◽  
Heat Stress ◽  
Real Time ◽  
Reference Genes ◽  
Dynamic Range ◽  
Expression Profiles ◽  
Mrna Levels ◽  
Expression Stability ◽  
Degenerate Primers ◽  
Different Tissues

Quantitative real-time reverse transcription PCR (RT-qPCR) has been widely used in the detection and quantification of gene expression levels because of its high accuracy, sensitivity, and reproducibility as well as its large dynamic range. However, the reliability and accuracy of RT-qPCR depends on accurate transcript normalization using stably expressed reference genes.Amorphophallusis a perennial plant with a high content of konjac glucomannan (KGM) in its corm. This crop has been used as a food source and as a traditional medicine for thousands of years. Without adequate knowledge of gene expression profiles, there has been no report of validated reference genes inAmorphophallus. In this study, nine genes that are usually used as reference genes in other crops were selected as candidate reference genes. These putative sequences of these genesAmorphophalluswere cloned by the use of degenerate primers. The expression stability of each gene was assessed in different tissues and under two abiotic stresses (heat and waterlogging) inA. albusandA. konjac. Three distinct algorithms were used to evaluate the expression stability of the candidate reference genes. The results demonstrated thatEF1-a,EIF4A,H3andUBQwere the best reference genes under heat stress inAmorphophallus. Furthermore,EF1-a,EIF4A,TUB, andRPwere the best reference genes in waterlogged conditions. By comparing different tissues from all samples, we determined thatEF1-α,EIF4A,andCYPwere stable in these sets. In addition, the suitability of these reference genes was confirmed by validating the expression of a gene encoding the small heat shock proteinSHSP, which is related to heat stress inAmorphophallus. In sum,EF1-αandEIF4Awere the two best reference genes for normalizing mRNA levels in different tissues and under various stress treatments, and we suggest using one of these genes in combination with 1 or 2 reference genes associated with different biological processes to normalize gene expression. Our results will provide researchers with appropriate reference genes for further gene expression quantification using RT-qPCR inAmorphophallus.

Multiplex qPCR assay for assessment of reference gene expression stability in rat tissues/samples

2020 ◽  
Vol 53 ◽  
pp. 101611 ◽  
Author(s):  
Alexander P. Schwarz ◽  
Daria A. Malygina ◽  
Anna A. Kovalenko ◽  
Alexander N. Trofimov ◽  
Aleksey V. Zaitsev
Keyword(s):  
Gene Expression ◽  
Reference Gene ◽  
Qpcr Assay ◽  
Rat Tissues ◽  
Expression Stability ◽  
Gene Expression Stability ◽  
Multiplex Qpcr ◽  
Reference Gene Expression

scholarly journals Hypoxanthine Guanine Phosphoribosyl Transferase Is the Most Stable Reference Gene for Gene Expression Analysis by Quantitative PCR in Peripheral Blood Mononuclear Cells from Women with the Polycystic Ovary Syndrome

2014 ◽  
Vol 33 (4) ◽  
pp. 356-363 ◽  
Author(s):  
Danijela Vojnović Milutinović ◽  
Djuro Macut ◽  
Ivana Božić Antić ◽  
Jelica Bjekić Macut ◽  
Marina Nikolić ◽  
...  
Keyword(s):  
Gene Expression ◽  
Reference Genes ◽  
Mononuclear Cells ◽  
Polycystic Ovary ◽  
Normal Weight ◽  
Expression Stability ◽  
Rt Pcr ◽  
Ovary Syndrome ◽  
Peripheral Blood Mononuclear ◽  
Software Packages

Summary Background: The polycystic ovary syndrome (PCOS) is a frequent endocrine disorder that affects women of reproductive age. As the syndrome is strongly associated with obesity, it is of interest to examine the gene expression diffe rences that accompany its development and the associ a ted metabolic disturbances. Real-time RT PCR is a standard method for studying changes in gene expression. However, to obtain accurate and reliable results, validation of reference genes is obligatory. The aim of this study was to identify a suitable reference for the normalization of gene expression in peripheral blood mononuclear cells (PBMCs) from obese and normal-weight women with PCOS. Methods: The expression stability of four potential reference genes: hypoxanthine guanine phosphoribosyl trans-ferase 1 (HPRT), β-actin (BA), β2-microglobulin (B2M) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH), was assessed in PBMCs from healthy women, and from normal-weight and obese women with PCOS. The variability in the expression of potential reference genes was analyzed by the TaqMan real-time RT PCR method, using GeNorm and NormFinder software packages. Results: Direct comparison of cycle threshold (Ct) values showed inter-individual variations for all validated genes, the Ct values of HPRT being less variable than those of BA, GAPDH and B2M. Both software packages pointed to HPRT as the most steadily expressed gene in the PBMCs of women with PCOS and healthy controls. Conclusions: Cross-validation of the expression stability of four potential reference genes identified HPRT as the most stable reference, suitable for further investigations of gene expression in PBMCs from women with PCOS.

Validation of reference genes for accurate normalization of gene expression for real time-quantitative PCR in strawberry fruits using different cultivars and osmotic stresses

Gene ◽  
2015 ◽  
Vol 554 (2) ◽  
pp. 205-214 ◽  
Author(s):  
Vanessa Galli ◽  
Joyce Moura Borowski ◽  
Ellen Cristina Perin ◽  
Rafael da Silva Messias ◽  
Julia Labonde ◽  
...  
Keyword(s):  
Gene Expression ◽  
Real Time ◽  
Quantitative Pcr ◽  
Reference Genes ◽  
Real Time Quantitative Pcr ◽  
Accurate Normalization

scholarly journals Establishing reference genes for use in real-time quantitative PCR analysis of early equine embryos

2011 ◽  
Vol 23 (2) ◽  
pp. 353 ◽  
Author(s):  
Damien B. B. P. Paris ◽  
Ewart W. Kuijk ◽  
Bernard A. J. Roelen ◽  
Tom A. E. Stout
Keyword(s):  
Gene Expression ◽  
Real Time ◽  
Quantitative Pcr ◽  
Embryo Development ◽  
Reference Genes ◽  
Stable Expression ◽  
Pcr Analysis ◽  
Blastocyst Development ◽  
Real Time Quantitative Pcr

Real-time quantitative PCR (qPCR) is invaluable for investigating changes in gene expression during early development, since it can be performed on the limited quantities of mRNA contained in individual embryos. However, the reliability of this method depends on the use of validated stably expressed reference genes for accurate data normalisation. The aim of the present study was to identify and validate a set of reference genes suitable for studying gene expression during equine embryo development. The stable expression of four carefully selected reference genes and one developmentally regulated gene was examined by qPCR in equine in vivo embryos from morula to expanded blastocyst stage. SRP14, RPL4 and PGK1 were identified by geNorm analysis as stably expressed reference genes suitable for data normalisation. RPL13A expression was less stable and changed significantly during the period of development examined, rendering it unsuitable as a reference gene. As anticipated, CDX2 expression increased significantly during embryo development, supporting its possible role in trophectoderm specification in the horse. In summary, it was demonstrated that evidence-based selection of potential reference genes can reduce the number needed to validate stable expression in an experimental system; this is particularly useful when dealing with tissues that yield small amounts of mRNA. SRP14, RPL4 and PGK1 are stable reference genes suitable for normalising expression for genes of interest during in vivo morula to expanded blastocyst development of horse embryos.

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