Selection and Validation of Appropriate Reference Genes for Real-Time Quantitative PCR Analysis in Needles of Larix olgensis under Abiotic Stresses

Larix olgensis Henry is an important afforestation species in northeastern China because of its fast juvenile growth, high-quality timber, and significant economic and ecological values. The selection of appropriate reference genes is necessary for the normalization of gene expression determination during quantitative real-time polymerase chain reaction (qRT-PCR) experiments. In this study, qRT-PCR was used to study gene expression. Three software packages geNorm, NormFinder, BestKeeper were used, and a comprehensive ranking of candidate reference genes was produced based on their output to evaluate the expression stability of 16 candidate reference genes from L. olgensis under drought, salt, cold, and heat stress. PP2A-1 and GAPDH ranked as the most stable reference genes under drought and cold stress, PP2A-1 and UBQ10 were most stable under salt stress, and TIP41 and ACT2 were most stable under heat stress. The least stable gene was ADP, which ranked the last under all treatments. Expression profile analysis of the antioxidant gene CAT using the two most stable and the single least stable reference genes under each stress further verified that the selected reference genes were suitable for gene expression normalization. This study provides an important foundation for the selection of suitable reference genes for the normalization and quantification of L. olgensis gene expression under abiotic stress conditions.

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Selection of Reliable Reference Genes for Real-time qRT-PCR Analysis of Zi Geese (<italic>Anser anser domestica</italic>) Gene Expression

Asian-Australasian Journal of Animal Sciences ◽

10.5713/ajas.2012.12417 ◽

2013 ◽

Vol 26 (3) ◽

pp. 423-432 ◽

Cited By ~ 6

Author(s):

Hong Ji ◽

Jianfa Wang ◽

Juxiong Liu ◽

Jingru Guo ◽

Zhongwei Wang ◽

...

Keyword(s):

Gene Expression ◽

Real Time ◽

Reference Genes ◽

Pcr Analysis ◽

Anser Anser ◽

Qrt Pcr ◽

Selection Of

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Selection of Reference Genes for qRT-PCR Analysis of Gene Expression in Stipa grandis during Environmental Stresses

PLoS ONE ◽

10.1371/journal.pone.0169465 ◽

2017 ◽

Vol 12 (1) ◽

pp. e0169465 ◽

Cited By ~ 6

Author(s):

Dongli Wan ◽

Yongqing Wan ◽

Qi Yang ◽

Bo Zou ◽

Weibo Ren ◽

...

Keyword(s):

Gene Expression ◽

Reference Genes ◽

Environmental Stresses ◽

Pcr Analysis ◽

Qrt Pcr ◽

Selection Of

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Selection of reference genes for qRT-PCR analysis of gene expression in sea cucumber Apostichopus japonicus during aestivation

Chinese Journal of Oceanology and Limnology ◽

10.1007/s00343-015-4004-2 ◽

2014 ◽

Vol 32 (6) ◽

pp. 1248-1256 ◽

Cited By ~ 9

Author(s):

Ye Zhao ◽

Muyan Chen ◽

Tianming Wang ◽

Lina Sun ◽

Dongxue Xu ◽

...

Keyword(s):

Gene Expression ◽

Reference Genes ◽

Sea Cucumber ◽

Apostichopus Japonicus ◽

Pcr Analysis ◽

Qrt Pcr ◽

Selection Of

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Selection of reference genes for quantitative real-time RT-PCR on gene expression in Golden Pompano (Trachinotus ovatus)

Polish Journal of Veterinary Sciences ◽

10.1515/pjvs-2017-0071 ◽

2017 ◽

Vol 20 (3) ◽

pp. 583-594 ◽

Cited By ~ 4

Author(s):

X.J. Chen ◽

X.Q. Zhang ◽

S. Huang ◽

Z.J. Cao ◽

Q.W. Qin ◽

...

Keyword(s):

Gene Expression ◽

Real Time ◽

Reference Genes ◽

Housekeeping Genes ◽

Poly I:C ◽

Stable Gene ◽

Trachinotus Ovatus ◽

Golden Pompano ◽

Normal Physiological Condition ◽

Selection Of

Abstract Golden pompano (Trachinotus ovatus) is an important economically fish species. In this study, with an aim to identify reliable reference genes for quantitative real-time PCR (qRT-PCR) in golden pompano, we evaluated the expression stability of eight housekeeping genes in the presence and absence of poly I:C stimulation in eight tissues. The PCR data was analyzed by geNorm and NormFinder algorithms. The results showed that the expression of all the examined genes exhibited tissue-dependent variations. When under normal physiological condition, geNorm and NormFinder identified B2M and 18S as suitable genes. When studying gene expression under conditions of poly I:C stimulation, the selection of the internal controls should be selected on a tissue basis. At 12 h stimulation, geNorm ranked Actin/UBCE, Actin/B2M, UBCE/B2M, Actin/UBCE, RPL13/B2M, UBCE/GAPDH, B2M/RPL13, and UBCE/B2M, respectively, as the most stably expressed genes in liver, spleen, kidney, gill, intestine, heart, muscle, and brain. Comparable ranking orders were produced by NormFinder. Similar results were obtained at 48 h stimulation. Taken together, these results indicate that B2M and 18S are the most stable gene across tissue types under normal physiological conditions. However, during poly I:C stimulation, no single gene or single pair of genes in the examined set of housekeeping genes can serve as a universal reference across all tissue types. If one gene is preferred, B2M, B2M, UBCE, Actin, B2M/RPL13, B2M, B2M, and RPL13 may be used in spleen, kidney, liver, gill, intestine, brain, muscle, and heart of golden pompano, respectively.

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Standardization of Gene Expression Quantification by Absolute Real-Time qRT-PCR System Using a Single Standard for Marker and Reference Genes

Biomarker Insights ◽

10.4137/bmi.s5596 ◽

2010 ◽

Vol 5 ◽

pp. BMI.S5596 ◽

Cited By ~ 7

Author(s):

Yi-Hong Zhou ◽

Vinay R. Raj ◽

Eric Siegel ◽

Liping Yu

Keyword(s):

Gene Expression ◽

Real Time ◽

Gene Expression Data ◽

Reference Genes ◽

Pcr Analysis ◽

Expression Data ◽

Internal Reference ◽

Expression Ratio ◽

Qrt Pcr ◽

Single Standard

In the last decade, genome-wide gene expression data has been collected from a large number of cancer specimens. In many studies utilizing either microarray-based or knowledge-based gene expression profiling, both the validation of candidate genes and the identification and inclusion of biomarkers in prognosis-modeling has employed real-time quantitative PCR on reverse transcribed mRNA (qRT-PCR) because of its inherent sensitivity and quantitative nature. In qRT-PCR data analysis, an internal reference gene is used to normalize the variation in input sample quantity. The relative quantification method used in current real-time qRT-PCR analysis fails to ensure data comparability pivotal in identification of prognostic biomarkers. By employing an absolute qRT-PCR system that uses a single standard for marker and reference genes (SSMR) to achieve absolute quantification, we showed that the normalized gene expression data is comparable and independent of variations in the quantities of sample as well as the standard used for generating standard curves. We compared two sets of normalized gene expression data with same histological diagnosis of brain tumor from two labs using relative and absolute real-time qRT-PCR. Base-10 logarithms of the gene expression ratio relative to ACTB were evaluated for statistical equivalence between tumors processed by two different labs. The results showed an approximate comparability for normalized gene expression quantified using a SSMR-based qRT-PCR. Incomparable results were seen for the gene expression data using relative real-time qRT-PCR, due to inequality in molar concentration of two standards for marker and reference genes. Overall results show that SSMR-based real-time qRT-PCR ensures comparability of gene expression data much needed in establishment of prognostic/predictive models for cancer patients–-a process that requires large sample sizes by combining independent sets of data.

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Evaluation and Selection of Appropriate Reference Genes for Real-Time Quantitative PCR Analysis of Gene Expression in Nile Tilapia (Oreochromis niloticus) during Vaccination and Infection

International Journal of Molecular Sciences ◽

10.3390/ijms16059998 ◽

2015 ◽

Vol 16 (12) ◽

pp. 9998-10015 ◽

Cited By ~ 22

Author(s):

Erlong Wang ◽

Kaiyu Wang ◽

Defang Chen ◽

Jun Wang ◽

Yang He ◽

...

Keyword(s):

Gene Expression ◽

Real Time ◽

Quantitative Pcr ◽

Oreochromis Niloticus ◽

Reference Genes ◽

Nile Tilapia ◽

Pcr Analysis ◽

Real Time Quantitative Pcr ◽

Nile Tilapia Oreochromis Niloticus ◽

Selection Of

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Selection of reference genes for real-time quantitative PCR analysis of gene expression in Glycyrrhiza glabra under drought stress

Biologia Plantarum ◽

10.1007/s10535-016-0601-y ◽

2016 ◽

Vol 60 (4) ◽

pp. 645-654 ◽

Cited By ~ 5

Author(s):

A. Maroufi

Keyword(s):

Gene Expression ◽

Drought Stress ◽

Real Time ◽

Quantitative Pcr ◽

Reference Genes ◽

Glycyrrhiza Glabra ◽

Pcr Analysis ◽

Real Time Quantitative Pcr ◽

Selection Of

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Reference Genes for Quantitative Real-Time PCR Analysis of Gene Expression in Mung Bean under Abiotic Stress and Cercospora canescens Infection

Legume Research - An International Journal ◽

10.18805/lr-507 ◽

2020 ◽

Author(s):

X. W. Ke ◽

L. H. Yin ◽

J. Xu ◽

W. N. Sun ◽

X. D. Xu ◽

...

Keyword(s):

Gene Expression ◽

Real Time ◽

Real Time Pcr ◽

Reference Genes ◽

Mung Bean ◽

Expression Profiles ◽

Pcr Analysis ◽

Saline Stress ◽

Stable Gene ◽

Quantitative Real Time Pcr

The objective of the study was to identify suitable reference genes that can be used for quantitative real-time PCR (qPCR) analysis in mung bean (Vigna radiata). Therefore, 10 potential reference genes were selected and the results showed that ubiquitin-conjugating enzyme was suitable as reference under drought and pathogen infection stress; elongation factor 1-á was the most stable gene under waterlogging; and actin performed the best under saline stress. These selected reference genes were further confirmed by analysis of the expression profiles of catalase and peroxidase under waterlogging. Our results will contribute to the improvement of the accuracy of gene expression evaluation in mung bean.

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

PeerJ ◽

10.7717/peerj.5226 ◽

2018 ◽

Vol 6 ◽

pp. e5226 ◽

Cited By ~ 6

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.

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