Screening for Suitable Reference Genes for Quantitative Real-Time PCR in Heterosigma akashiwo (Raphidophyceae)

Quantitative real-time PCR (qPCR) is widely used in molecular biology, although the accuracy of the quantitative results is determined by the stability of the reference genes used. Recent studies have investigated suitable reference genes for some crustaceans under various conditions, but studies in Macrobrachium nipponense are currently lacking. In this study, we selected the following seven genes from among 35 commonly used housekeeping genes as candidate qPCR reference genes for temporal and spatial expression: EIF (eukaryotic translation initiation factor 5A), 18S (18S ribosomal RNA), EF-1α (elongation factor-1α), GAPDH (glyceraldehyde-3-phosphate dehydrogenase), TUB (α-tubulin), β-act (β-actin), and RPL18 (Ribosomal protein L18). The stability of each reference gene was evaluated by GeNorm, NormFinder, BestKeeper, and comparative ∆C t methods, and was comprehensively ranked using RefFinder. RPL18 was shown to be the most suitable reference gene for adult M. nipponense tissues, while EIF was the most stable in different ovarian and embryo stages and in white spot syndrome virus infection, and β-act was the most stable reference gene under hypoxia stress. The reliability of the rankings was confirmed by RNA interference experiments. To the best of our knowledge, this represents the first systematic analysis of reference genes for qPCR experiments in M. nipponense, and the results will provide invaluable information for future research in closely related crustaceans.

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Selection of Suitable Reference Genes for Quantitative Real-Time PCR Normalization in Human Stem Cell Research

Advances in Experimental Medicine and Biology - Cell Biology and Translational Medicine, Volume 4 ◽

10.1007/5584_2018_277 ◽

2018 ◽

pp. 151-168 ◽

Cited By ~ 1

Author(s):

Fatma Betül Ayanoğlu ◽

Ayşe Eser Elçin ◽

Yaşar Murat Elçin

Keyword(s):

Stem Cell ◽

Real Time ◽

Stem Cell Research ◽

Real Time Pcr ◽

Reference Genes ◽

Human Stem Cell ◽

Quantitative Real Time Pcr ◽

Suitable Reference ◽

Selection Of

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Selection of suitable reference genes for reverse transcription quantitative real-time PCR studies on different experimental systems from carrot (Daucus carota L.)

Scientia Horticulturae ◽

10.1016/j.scienta.2014.12.038 ◽

2015 ◽

Vol 186 ◽

pp. 115-123 ◽

Cited By ~ 18

Author(s):

Maria Doroteia Campos ◽

António Miguel Frederico ◽

Thomas Nothnagel ◽

Birgit Arnholdt-Schmitt ◽

Hélia Cardoso

Keyword(s):

Real Time ◽

Reverse Transcription ◽

Daucus Carota ◽

Real Time Pcr ◽

Reference Genes ◽

Quantitative Real Time Pcr ◽

Daucus Carota L ◽

Experimental Systems ◽

Suitable Reference ◽

Selection Of

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Selection of Suitable Reference Genes for Quantitative Real-time PCR in Sapium sebiferum

Frontiers in Plant Science ◽

10.3389/fpls.2017.00637 ◽

2017 ◽

Vol 8 ◽

Cited By ~ 10

Author(s):

Xue Chen ◽

Yingji Mao ◽

Shengwei Huang ◽

Jun Ni ◽

Weili Lu ◽

...

Keyword(s):

Real Time ◽

Real Time Pcr ◽

Reference Genes ◽

Sapium Sebiferum ◽

Quantitative Real Time Pcr ◽

Suitable Reference ◽

Selection Of

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Selection of appropriate reference genes for quantitative real-time PCR in Clerodendrum trichotomum

10.1101/625145 ◽

2019 ◽

Author(s):

Yajie Hua ◽

Yuanzheng Yue ◽

Gongwei Chen ◽

Taotao Yan ◽

Wenjie Ding ◽

...

Keyword(s):

Real Time ◽

Real Time Pcr ◽

Reference Genes ◽

Gene Expression Analysis ◽

Quantitative Real Time Pcr ◽

Mechanism Research ◽

The Stability ◽

Suitable Reference ◽

Important Medicinal Plant ◽

Selection Of

AbstrsctClerodendrum trichotomum, an important medicinal plant, has excellent salt tolerance and beautiful ornamental character. However, reliable reference genes for quantitative real-time PCR data (qRT-PCR) in C. trichotomum have not been investigated. Using our previous transcriptome data, 17 reference genes were selected in different tissues (leaves, flowers, fruits, stems, and roots) and under various abiotic stresses (salt, drought, flood, and heat) for C. trichotomum, using four different reference gene analysis software types: GeNorm, NormFinder, BestKeeper and ReFinder. The results identified RPL, ACT and HSP70 as the three most suitable reference genes for tissues. Genes ACT and AP-2 were most stably expressed under drought stress; MDH and UBCE2 were stable under flooding stress; RPL and UBCE2 were most stable under salt stress; and MDH and EF-1A were most appropriate under heat stress. For abiotic treatments, RPL, MDH and AP-2 were the most stable reference genes; and AP-2, RPL and ACT were stably expressed in all examined samples. The expression profile of the genes for Na+/H+ Exchanger1 (ClNHX1) and laccase (ClLAC) were selected to validate the stability of the determined reference genes. Our study provided reliable normalization for gene expression analysis and ensured more accurate data for further molecular mechanism research in C. trichotomum.

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Systematic selection and validation of suitable reference genes for quantitative real-time PCR normalization studies of gene expression in Nitraria tangutorum

Scientific Reports ◽

10.1038/s41598-020-73059-3 ◽

2020 ◽

Vol 10 (1) ◽

Author(s):

Bo Wang ◽

Huirong Duan ◽

Peifang Chong ◽

Shiping Su ◽

Lishan Shan ◽

...

Keyword(s):

Gene Expression ◽

Real Time ◽

Real Time Pcr ◽

Reference Genes ◽

Expression Stability ◽

Quantitative Real Time Pcr ◽

Experimental Conditions ◽

Nitraria Tangutorum ◽

Multiple Species ◽

Suitable Reference

Abstract Suitable reference genes can be used to calibrate the error in quantitative real-time PCR (qPCR) experiments, making the results more credible. However, there are no reference genes suitable for multiple species and under different experimental conditions. Nitraria tangutorum Bobr. is a typical plant native to desert areas. It is drought-resistant, saline-alkali resistant, extreme temperatures-resistant, and has strong adaptability. To date, the importance of this germplasm has not been sufficiently understood; therefore, it is still unclear which genes can be used as reference genes to calibrate qPCR data of N. tangutorum. In this study we analyzed the expression levels of 10 candidate reference genes (ACT, GAPDH, TUA, TUB, CYP, UBC, His, PP2A, HSP, and EF1-α) in N. tangutorum seedlings under a series of experimental conditions, including in different organs (root, stem, and leaf) and under abiotic stresses (salt, drought, heat, and cold) and hormone stimuli (abscisic acid) by qPCR. Three software programs (geNorm, NormFinder, and BestKeeper) were used to evaluate the expression stability of the ten genes. Comprehensive analysis showed that EF1-α and His had the best expression stability, whereas HSP was the least suitable as a reference gene. The expression profile of NtCER7, a gene related to the regulation of cuticular wax biosynthesis in N. tangutorum, verified the accuracy of the experimental results. Based on this study, we recommend EF1-α and His as suitable reference genes for N. tangutorum. This paper provides the first data on stable reference genes in N. tangutorum, which will be beneficial to studying the gene expression of N. tangutorum and other Nitraria species in the future.

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