Identification of candidate reference genes for qRT-PCR normalization studies of salinity stress and injury in Onchidium reevesii

Real-time quantitative reverse transcription-PCR (qRT-PCR) is an undeniably effective tool for measuring levels of gene expression, but the accuracy and reliability of the statistical data obtained depend mainly on the basal expression of selected housekeeping genes in many samples. To date, there have been few analyses of stable housekeeping genes in Onchidium reevesii under salinity stress and injury. In this study, the gene expression stabilities of seven commonly used housekeeping genes, CYC, RPL28S, ACTB, TUBB, EF1a, Ubiq and 18S RNA, were investigated using BestKeeper, geNorm, NormFinder and RefFinfer. Although the results of the four programs varied to some extent, in general, RPL28S, TUBB, ACTB and EF1a were ranked highly. ACTB and TUBB were found to be the most stable housekeeping genes under salinity stress, and EF1a plus TUBB was the most stable combination under injury stress. When analysing target gene expression in different tissues, RPL28S or EF1a should be selected as the reference gene according to the level of target gene expression. Under extreme environmental stress (salinity) conditions, ACTB (0 ppt, 5 ppt, 15 ppt, 25 ppt) and TUBB (35 ppt) are reasonable reference gene choices when expression stability and abundance are considered. Under conditions of 15 ppt salinity and injury stress, our results showed that the best two-gene combination was TUBB plus EF1a. Therefore, we suggest that RPL28S, ACTB and TUBB are suitable reference genes for evaluating mRNA transcript levels. Based on candidate gene expression analysis, the tolerance of O. reevesii to low salinity (low osmotic pressure) is reduced compared to its tolerance to high salinity (high osmotic pressure). These findings will help researchers obtain accurate results in future quantitative gene expression analyses of O. reevesii under other stress conditions.

Download Full-text

Validation of internal reference genes for relative quantitation studies of gene expression in human laryngeal cancer

PeerJ ◽

10.7717/peerj.2763 ◽

2016 ◽

Vol 4 ◽

pp. e2763 ◽

Cited By ~ 3

Author(s):

Xiaofeng Wang ◽

Jinting He ◽

Wei Wang ◽

Ming Ren ◽

Sujie Gao ◽

...

Keyword(s):

Gene Expression ◽

Reference Gene ◽

Laryngeal Cancer ◽

Reference Genes ◽

Target Gene ◽

Cancer Tissue ◽

Internal Reference ◽

Target Gene Expression ◽

Internal Reference Gene ◽

Relative Quantitation

BackgroundThe aim of this study was to determine the expression stabilities of 12 common internal reference genes for the relative quantitation analysis of target gene expression performed by reverse transcription real-time quantitative polymerase chain reaction (RT-qPCR) in human laryngeal cancer.MethodsHep-2 cells and 14 laryngeal cancer tissue samples were investigated. The expression characteristics of 12 internal reference gene candidates (18S rRNA, GAPDH, ACTB, HPRT1, RPL29, HMBS, PPIA, ALAS1, TBP, PUM1, GUSB, and B2M) were assessed by RT-qPCR. The data were analyzed by three commonly used software programs: geNorm, NormFinder, and BestKeeper.ResultsThe use of the combination of four internal reference genes was more appropriate than the use of a single internal reference gene. The optimal combination was PPIA + GUSB + RPL29 + HPRT1 for both the cell line and tissues; while the most appropriate combination was GUSB + RPL29 + HPRT1 + HMBS for the tissues.ConclusionsOur recommended internal reference genes may improve the accuracy of relative quantitation analysis of target gene expression performed by the RT-qPCR method in further gene expression research on laryngeal tumors.

Download Full-text

Identification and Evaluation of Reference Genes for Normalization of Gene Expression in Developmental Stages, Sexes, and Tissues of Diaphania caesalis (Lepidoptera, Pyralidae)

Journal of Insect Science ◽

10.1093/jisesa/iez130 ◽

2020 ◽

Vol 20 (1) ◽

Cited By ~ 2

Author(s):

Zheng Wang ◽

Qianqian Meng ◽

Xi Zhu ◽

Shiwei Sun ◽

Aiqin Liu ◽

...

Keyword(s):

Gene Expression ◽

Reference Genes ◽

Target Gene ◽

Target Genes ◽

Developmental Stages ◽

Expression Profiles ◽

Transcriptional Level ◽

Internal Reference ◽

Target Gene Expression ◽

Qrt Pcr

Abstract Diaphania caesalis (Walker) is an important boring insect mainly distributed in subtropical and tropical areas and attacked tropical woody grain crops, such as starchy plants of Artocarpus. Quantitative real-time polymerase chain reaction (qRT-PCR) is a powerful approach for investigating target genes expression profiles at the transcriptional level. However, the identification and selection of internal reference genes, which is often overlooked, is the most vital step before the analysis of target gene expression by qRT-PCR. So far, the reliable internal reference genes under a certain condition of D. caesalis have not been investigated. Therefore, this study evaluated the expression stability of eight candidate reference genes including ACT, β-TUB, GAPDH, G6PDH, RPS3a, RPL13a, EF1α, and EIF4A in different developmental stages, tissues and sexes using geNorm, NormFinder and BestKeeper algorithms. To verify the stability of the recommended internal reference genes, the expression levels of DcaeOBP5 were analyzed under different treatment conditions. The results indicated that ACT, RPL13a, β-TUB, RPS3a, and EF1α were identified as the most stable reference genes for further studies on target gene expression involving different developmental stages of D. caesalis. And ACT and EIF4A were recommended as stable reference genes for different tissues. Furthermore, ACT, EF1α, and RPS3a were ranked as the best reference genes in different sexes based on three algorithms. Our research represents the critical first step to normalize qRT-PCR data and ensure the accuracy of expression of target genes involved in phylogenetic and physiological mechanism at the transcriptional level in D. caesalia.

Download Full-text

Reference gene selection for qRT-PCR analyses of luffa (Luffa cylindrica) plants under abiotic stress conditions

Scientific Reports ◽

10.1038/s41598-021-81524-w ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Min-dong Chen ◽

Bin Wang ◽

Yong-ping Li ◽

Mei-juan Zeng ◽

Jian-ting Liu ◽

...

Keyword(s):

Gene Expression ◽

Reference Gene ◽

Reference Genes ◽

Gene Selection ◽

Expression Patterns ◽

Suitable Reference Gene ◽

Expression Levels ◽

Qrt Pcr ◽

Suitable Reference ◽

Gene Expression Levels

AbstractSelecting suitable internal reference genes is an important prerequisite for the application of quantitative real-time PCR (qRT-PCR). However, no systematic studies have been conducted on reference genes in luffa. In this study, seven reference genes were selected, and their expression levels in luffa plants exposed to various simulated abiotic stresses [i.e., cold, drought, heat, salt, H2O2, and abscisic acid (ABA) treatments] were analyzed by qRT-PCR. The stability of the reference gene expression levels was validated using the geNorm, NormFinder, BestKeeper, and RefFinder algorithms. The results indicated that EF-1α was the most stably expressed and suitable reference gene overall and for the heat, cold, and ABA treatments. Additionally, UBQ expression was stable following the salt treatment, whereas TUB was identified as a suitable reference gene for H2O2 and drought treatments. The reliability of the selected reference genes was verified by analyzing the expression of copper/zinc superoxide dismutase (Cu/Zn-SOD) gene in luffa. When the most unstable reference genes were used for data normalizations, the resulting expression patterns had obvious biases when compared with the expression patterns for the most ideal reference genes used alone or combined. These results will be conducive to more accurate quantification of gene expression levels in luffa.

Download Full-text

Reference Gene Selection for qRT-PCR Analyses of Luffa (Luffa cylindrica) Plants Under Abiotic Stress Conditions

10.21203/rs.3.rs-65316/v1 ◽

2020 ◽

Author(s):

mindong chen ◽

bin wang ◽

yongping li ◽

meijuan zeng ◽

jianting liu ◽

...

Keyword(s):

Gene Expression ◽

Reference Gene ◽

Abiotic Stresses ◽

Reference Genes ◽

Expression Patterns ◽

Cold Treatment ◽

Suitable Reference Gene ◽

Expression Levels ◽

Qrt Pcr ◽

Suitable Reference

Abstract Background: Quantitative real-time PCR (qRT-PCR) is one of the preferred methods for analyzing gene expression, and selecting suitable internal reference genes is an important prerequisite for the application of this technology. However, no systematic studies have been conducted on reference genes in luffa, resulting in limited investigations of luffa gene expression. Results: In this study, seven reference genes ( ACT , TUA , TUB , EF-1α , GAPDH , UBQ , and 18S ) were selected, and their expression levels in luffa plants exposed to various simulated abiotic stresses [i.e., cold, drought, heat, salt, H 2 O 2 , and abscisic acid (ABA) treatments] were analyzed by qRT-PCR. The stability of the reference gene expression levels was validated using the geNorm, NormFinder, BestKeeper, and RefFinder algorithms. The results indicated that EF-1α was the most stably expressed and suitable reference gene overall and for the heat, cold, and ABA treatments. Additionally, UBQ expression was stable following the salt treatment, whereas TUB was identified as a suitable reference gene for H 2 O 2 and drought treatments. In contrast, GAPDH was revealed as an unsuitable reference gene overall and for the heat, salt, H 2 O 2 , ABA, and drought treatments. Regarding the cold treatment, TUA was identified as an unsuitable reference gene. The reliability of the selected reference genes was verified by analyzing the expression of copper/zinc superoxide dismutase ( Cu/Zn-SOD ) gene in luffa. When the most unstable reference genes were used for data normalizations, the resulting expression patterns had obvious biases when compared with the expression patterns for the most ideal reference genes used alone or combined. Conclusions: The study data were used to compile a list of suitable reference genes for qRT-PCR analyses of the gene expression in luffa plants exposed to abiotic stresses. This work may provide the basis for future qRT-PCR-based investigations of the transcription of important functional genes in luffa.

Download Full-text

Transcriptome-wide identification and validation of reference genes of Black rockfish (Sebastes schlegelii) for quantitative RT-PCR

10.21203/rs.3.rs-17096/v1 ◽

2020 ◽

Author(s):

Chaofan Jin ◽

Weihao Song ◽

Mengya Wang ◽

Jie Qi ◽

Quanqi Zhang ◽

...

Keyword(s):

Gene Expression ◽

Reference Gene ◽

Reference Genes ◽

Developmental Stages ◽

Suitable Reference Gene ◽

Rt Pcr ◽

Black Rockfish ◽

Qrt Pcr ◽

Sebastes Schlegelii ◽

Suitable Reference

Abstract Background: The quantitative real-time reverse transcription PCR (qRT-PCR) is a widely used technique that relies on the reference gene for gene expression normalization. Selecting a suitable reference gene is a crucial step to obtain an accurate result in qRT-PCR. However, most previous studies of fishes adopted reference genes that were commonly used in mammals without validation. Results: In this study, we utilized 89 transcriptome datasets covering early developmental stages and adult tissues, and carried out transcriptome-wide identification and validation of reference genes in Sebastes schlegelii. Finally, 121 candidate reference genes were identified based on four criteria. Eight candidates (METAP2, BTF3L4, EIF5A1, TCTP, UBC, PAIRB, RAB10, and DLD) and four commonly used reference genes (TUBA, ACTB, GAPDH, RPL17) in mammals were selected for validation via qRT- PCR and four statistical methods (delta-Ct, BestKeeper, geNorm, and NormFinder). The results revealed that the candidate reference genes we recommended are more stable than traditionally used ones. Conclusions: This is the first study to conduct transcriptome-wide identification and validation of reference genes for quantitative RT-PCR in the black rockfish, and lay an important foundation for gene expression analysis in teleost.

Download Full-text

Identification and selection of reference genes for gene expression analysis by quantitative real-time PCR in Suaeda glauca’s response to salinity

Scientific Reports ◽

10.1038/s41598-021-88151-5 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Meng Wang ◽

Tingting Ren ◽

Prince Marowa ◽

Haina Du ◽

Zongchang Xu

Keyword(s):

Gene Expression ◽

Real Time ◽

Reference Gene ◽

Reference Genes ◽

Suitable Reference Gene ◽

Normalize Gene Expression ◽

Suaeda Glauca ◽

Suitable Reference ◽

Germinating Seeds ◽

Different Tissues

AbstractQuantitative real-time polymerase chain reaction (qPCR) using a stable reference gene is widely used for gene expression research. Suaeda glauca L. is a succulent halophyte and medicinal plant that is extensively used for phytoremediation and extraction of medicinal compounds. It thrives under high-salt conditions, which promote the accumulation of high-value secondary metabolites. However, a suitable reference gene has not been identified for gene expression standardization in S. glauca under saline conditions. Here, 10 candidate reference genes, ACT7, ACT11, CCD1, TUA5, UPL1, PP2A, DREB1D, V-H+-ATPase, MPK6, and PHT4;5, were selected from S. glauca transcriptome data. Five statistical algorithms (ΔCq, geNorm, NormFinder, BestKeeper, and RefFinder) were applied to determine the expression stabilities of these genes in 72 samples at different salt concentrations in different tissues. PP2A and TUA5 were the most stable reference genes in different tissues and salt treatments, whereas DREB1D was the least stable. The two reference genes were sufficient to normalize gene expression across all sample sets. The suitability of identified reference genes was validated with MYB and AP2 in germinating seeds of S. glauca exposed to different NaCl concentrations. Our study provides a foundational framework for standardizing qPCR analyses, enabling accurate gene expression profiling in S. glauca.

Download Full-text

Identification of the most suitable reference gene for gene expression studies with development and abiotic stress response in Bromus sterilis

Scientific Reports ◽

10.1038/s41598-021-92780-1 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Madhab Kumar Sen ◽

Kateřina Hamouzová ◽

Pavlina Košnarová ◽

Amit Roy ◽

Josef Soukup

Keyword(s):

Reference Gene ◽

Herbicide Resistance ◽

Reference Genes ◽

Gene Selection ◽

High Yield ◽

Suitable Reference Gene ◽

Grass Species ◽

Experimental Conditions ◽

Qrt Pcr ◽

Suitable Reference

AbstractBromus sterilis is an annual weedy grass, causing high yield losses in winter cereals. Frequent use of herbicides had led to the evolution of herbicide resistance in this species. Mechanisms underlying herbicide resistance in B. sterilis must be uncovered because this problem is becoming a global threat. qRT-PCR and the next-generation sequencing technologies can elucidate the resistance mechanisms. Although qRT-PCR can calculate precise fold changes, its preciseness depends on the expression of reference genes. Regardless of stable expression in any given condition, no gene can act as a universal reference gene. Hence, it is necessary to identify the suitable reference gene for each species. To our knowledge, there are no reports on the suitable reference gene in any brome species so far. Thus, in this paper, the stability of eight genes was evaluated using qRT-PCR experiments followed by expression stability ranking via five most commonly used software for reference gene selection. Our findings suggest using a combination of 18S rRNA and ACCase to normalise the qRT-PCR data in B. sterilis. Besides, reference genes are also recommended for different experimental conditions. The present study outcomes will facilitate future molecular work in B. sterilis and other related grass species.

Download Full-text

Identification and validation of miRNA reference genes in poplar under pathogen stress

10.21203/rs.3.rs-225734/v1 ◽

2021 ◽

Author(s):

Lichun Zhang ◽

Xiaoqian Yang ◽

Yiyi Yin ◽

Jinxing Wang ◽

Yanwei Wang

Keyword(s):

Gene Expression ◽

Reference Gene ◽

Small Rna ◽

Reference Genes ◽

Expression Profiles ◽

Small Rna Sequencing ◽

Rna Seq ◽

Internal Standards ◽

Qrt Pcr ◽

Canker Pathogen

Abstract Quantitative real time polymerase chain reaction (qRT-PCR) is a common method to analyze gene expression. Due to differences in RNA quantity, quality, and reverse transcription efficiency between qRT-PCR samples, reference genes are used as internal standards to normalize gene expression. However, few universal genes especially miRNAs have been identified as reference so far. Therefore, it is essential to identify reference genes that can be used across various experimental conditions, stress treatments, or tissues. In this study, 14 microRNAs (miRNAs) and 5.8S rRNA were assessed for expression stability in poplar trees infected with canker pathogen. Using three reference gene analysis programs, we found that miR156g and miR156a exhibited stable expression throughout the infection process. miR156g and miR156a were then tested as internal standards to measure the expression of miR1447 and miR171c, and the results were compared to small RNA sequencing (RNA-seq) data. We found that when miR156a was used as the reference gene, the expression of miR1447 and miR171c were consistent with the small RNA-seq expression profiles. Therefore, miR156a was the most stable miRNAs examined in this study, and could be used as a reference gene in poplar under canker pathogen stress, which should enable comprehensive comparisons of miRNAs expression and avoid the bias caused by different lenth between detected miRNAs and traditional referece genes. The present study has expanded the miRNA reference genes available for gene expression studies in trees under biotic stress.

Download Full-text

Selecting Suitable Reference Genes for qPCR Normalization: A Comprehensive Analysis in MCF-7 Breast Cancer Cell Line

10.21203/rs.3.rs-42293/v2 ◽

2020 ◽

Author(s):

Nityanand Jain ◽

Dina Nitisa ◽

Valdis Pirsko ◽

Inese Cakstina

Keyword(s):

Gene Expression ◽

Cell Line ◽

Reference Gene ◽

Reference Genes ◽

Cancer Cell Line ◽

Internal Controls ◽

Reference Gene Expression ◽

Qpcr Normalization ◽

Suitable Reference ◽

Mcf 7

Abstract BackgroundMCF-7 breast cancer cell line is undoubtedly amongst the most extensively studied patient-derived research models, providing pivotal results that have over the decades translated to constantly improving patient care. Many research groups, have previously identified suitable reference genes for qPCR normalization in MCF-7 cell line. However, over the course of identification of suitable reference genes, a comparative analysis comprising these genes together in a single study have not been reported. Furthermore, the expression dynamics of these reference genes within sub-clones cultured over multiple passages (p) has attracted limited attention from research groups. Therefore, we investigated the expression dynamics of 12 previously suggested reference genes within two sub-clones (culture A1 and A2) cultured identically over multiple passages. Additionally, the effect of nutrient stress on reference gene expression was examined to devise an evidence-based recommendation of the least variable reference genes that could be employed in future gene expression studies.ResultsThe analysis revealed the presence of differential reference gene expression within the sub-clones of MCF-7. In culture A1, GAPDH-CCSER2 were identified as the least variable reference gene pair while for culture A2, GAPDH-RNA28S was identified. However, upon validation using genes of interest, both these pairs were found to be unsuitable control pairs. Normalization of AURKA and KRT19 with triplet pair GAPDH-PCBP1-CCSER2 yielded successful results. The triplet also proved its capability to handle variations arising from nutrient stress.ConclusionsThe variance in expression behavior amongst sub-clones highlights the potential need for exercising caution while selecting reference genes for MCF-7. GAPDH-PCBP1-CCSER2 triplet offers a reliable alternative to otherwise traditionally used internal controls for optimizing intra- and inter-assay gene expression differences. Furthermore, we suggest avoiding the use of ACTB, GAPDH and PGK1 as single internal controls.

Download Full-text

Comparison of Reliable Reference Genes Following Different Hormone Treatments by Various Algorithms for qRT-PCR Analysis of Metasequoia

International Journal of Molecular Sciences ◽

10.3390/ijms20010034 ◽

2018 ◽

Vol 20 (1) ◽

pp. 34 ◽

Cited By ~ 3

Author(s):

Jing-Jing Wang ◽

Shuo Han ◽

Weilun Yin ◽

Xinli Xia ◽

Chao Liu

Keyword(s):

Gene Expression ◽

Reference Genes ◽

Elongation Factor ◽

Pcr Analysis ◽

Qrt Pcr ◽

Gene Normalization ◽

Accurate Normalization ◽

Suitable Reference ◽

Different Tissues ◽

Gene Expression Levels

Quantitative reverse transcription polymerase chain reaction (qRT-PCR) is the most sensitive technique for evaluating gene expression levels. Choosing appropriate reference genes for normalizing target gene expression is important for verifying expression changes. Metasequoia is a high-quality and economically important wood species. However, few systematic studies have examined reference genes in Metasequoia. Here, the expression stability of 14 candidate reference genes in different tissues and following different hormone treatments were analyzed using six algorithms. Candidate reference genes were used to normalize the expression pattern of FLOWERING LOCUS T and pyrabactin resistance-like 8. Analysis using the GrayNorm algorithm showed that ACT2 (Actin 2), HIS (histone superfamily protein H3) and TATA (TATA binding protein) were stably expressed in different tissues. ACT2, EF1α (elongation factor-1 alpha) and HIS were optimal for leaves treated with the flowering induction hormone solution, while Cpn60β (60-kDa chaperonin β-subunit), GAPDH (glyceraldehyde-3-phosphate dehydrogenase) and HIS were the best reference genes for treated buds. EF1α, HIS and TATA were useful reference genes for accurate normalization in abscisic acid-response signaling. Our results emphasize the importance of validating reference genes for qRT-PCR analysis in Metasequoia. To avoid errors, suitable reference genes should be used for different tissues and hormone treatments to increase normalization accuracy. Our study provides a foundation for reference gene normalization when analyzing gene expression in Metasequoia.

Download Full-text