Selection and validation of reference genes for quantitative gene expression normalization in Taxus spp.

AbstractQuantitative real-time PCR (qRT-PCR) is commonly used to measure gene expression to further explore gene function, while suitable reference genes must be stably expressed under different experimental conditions to obtain accurate and reproducible data for relative quantification. Taxol or paclitaxel is an important anticancer compound mainly identified in Taxus spp. The molecular mechanism of the regulation of taxol biosynthesis is current research goal. However, in the case of Taxus spp., few reports were published on screening suitable reference genes as internal controls for qRT-PCR. Here, eight reference genes were selected as candidate reference genes for further study. Common statistical algorithms geNorm, NormFinder, BestKeeper, ΔCt, and RefFinder were used to analyze the data from samples collected from a cell line of Taxus × media under various experimental conditions and from tissues of Taxus chinensis var. mairei. The expression patterns of TcMYC under salicylic acid treatment differed significantly, with the best and worst reference genes in the cell line. This study screened out suitable reference genes (GAPDH1 and SAND) under different treatments and tissues for the accurate and reliable normalization of the qRT-PCR expression data of Taxus spp. At the same time, this study will aid future research on taxol biosynthesis-related genes expression in Taxus spp., and can also be directly used to other related species.

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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.

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Selection and validation of reference genes for normalization of qRT-PCR data to study the cannabinoid pathway genes in industrial hemp

PLoS ONE ◽

10.1371/journal.pone.0260660 ◽

2021 ◽

Vol 16 (12) ◽

pp. e0260660

Author(s):

Michihito Deguchi ◽

Shobha Potlakayala ◽

Zachary Spuhler ◽

Hannah George ◽

Vijay Sheri ◽

...

Keyword(s):

Gene Expression ◽

Heavy Metal ◽

Reference Genes ◽

Expression Patterns ◽

Heavy Metal Stress ◽

Future Research ◽

Qrt Pcr ◽

Differential Gene ◽

Expression Characterization ◽

Industrial Hemp

There has been significant interest in researching the pharmaceutical applications of Industrial hemp since its legalization three years ago. The crop is mostly dioecious and known for its production of phytocannabinoids, flavonoids, and terpenes. Although many scientific reports have showed gene expression analysis of hemp through OMICs approaches, unreliable reference genes for normalization of qRT-PCR data make it difficult to validate the OMICs data. Four software packages: geNorm, NormFinder, BestKeeper, and RefFinder were used to evaluate the differential gene expression patterns of 13 candidate reference genes under osmotic, heavy metal, hormonal, and UV stresses. EF-1α ranked as the most stable reference gene across all stresses, TUB was the most stable under osmotic stress, and TATA was the most stable under both heavy metal stress and hormonal stimuli. The expression patterns of two cannabinoid pathway genes, AAE1 and CBDAS, were used to validate the reliability of the selected reference genes. This work provides useful information for gene expression characterization in hemp and future research in the synthesis, transport, and accumulation of secondary metabolites.

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Selection of Reference Genes for Quantitative Gene Expression in Porcine Mesenchymal Stem Cells Derived from Various Sources along with Differentiation into Multilineages

Stem Cells International ◽

10.1155/2015/235192 ◽

2015 ◽

Vol 2015 ◽

pp. 1-14 ◽

Cited By ~ 7

Author(s):

Won-Jae Lee ◽

Ryoung-Hoon Jeon ◽

Si-Jung Jang ◽

Ji-Sung Park ◽

Seung-Chan Lee ◽

...

Keyword(s):

Gene Expression ◽

Reference Genes ◽

Optimal Number ◽

Experimental Conditions ◽

Pearson’S Correlation ◽

Qrt Pcr ◽

Differentiated Cells ◽

Pearson's Correlation ◽

Suitable Reference

The identification of stable reference genes is a prerequisite for ensuring accurate validation of gene expression, yet too little is known about stable reference genes of porcine MSCs. The present study was, therefore, conducted to assess the stability of reference genes in porcine MSCs derived from bone marrow (BMSCs), adipose (AMSCs), and skin (SMSCs) with their in vitro differentiated cells into mesenchymal lineages such as adipocytes, osteocytes, and chondrocytes. Twelve commonly used reference genes were investigated for their threshold cycle (Ct) values by qRT-PCR. The Ct values of candidate reference genes were analyzed by geNorm software to clarify stable expression regardless of experimental conditions. Thus, Pearson’s correlation was applied to determine correlation between the three most stable reference genes (NF3) and optimal number of reference genes (NFopt). In assessment of stability of reference gene across experimental conditions by geNorm analysis, undifferentiated MSCs and each differentiated status into mesenchymal lineages showed slightly different results but similar patterns about more or less stable rankings. Furthermore, Pearson’s correlation revealed high correlation (r>0.9) between NF3and NFopt. Overall, the present study showed thatHMBS,YWHAZ,SDHA, andTBPare suitable reference genes for qRT-PCR in porcine MSCs.

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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.

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Validation of Reference Genes for Studying Different Abiotic Stresses in oat (Avena sativa L.) by RT-qPCR

Plants ◽

10.3390/plants10071272 ◽

2021 ◽

Vol 10 (7) ◽

pp. 1272

Author(s):

Judit Tajti ◽

Magda Pál ◽

Tibor Janda

Keyword(s):

Gene Expression ◽

Avena Sativa ◽

Abiotic Stresses ◽

Reference Genes ◽

Nutritional Value ◽

Tissue Type ◽

Future Research ◽

Experimental Conditions ◽

Expression Studies ◽

Gene Expression Studies

Oat (Avena sativa L.) is a widely cultivated cereal with high nutritional value and it is grown mainly in temperate regions. The number of studies dealing with gene expression changes in oat continues to increase, and to obtain reliable RT-qPCR results it is essential to establish and use reference genes with the least possible influence caused by experimental conditions. However, no detailed study has been conducted on reference genes in different tissues of oat under diverse abiotic stress conditions. In our work, nine candidate reference genes (ACT, TUB, CYP, GAPD, UBC, EF1, TBP, ADPR, PGD) were chosen and analysed by four statistical methods (GeNorm, Normfinder, BestKeeper, RefFinder). Samples were taken from two tissues (leaves and roots) of 13-day-old oat plants exposed to five abiotic stresses (drought, salt, heavy metal, low and high temperatures). ADPR was the top-rated reference gene for all samples, while different genes proved to be the most stable depending on tissue type and treatment combinations. TUB and EF1 were most affected by the treatments in general. Validation of reference genes was carried out by PAL expression analysis, which further confirmed their reliability. These results can contribute to reliable gene expression studies for future research in cultivated oat.

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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.

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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.

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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.

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Expression Analysis of XTH in Stem Swelling of Stem Mustard and Selection of Reference Genes

Genes ◽

10.3390/genes11010113 ◽

2020 ◽

Vol 11 (1) ◽

pp. 113 ◽

Cited By ~ 4

Author(s):

Mengyao Li ◽

Fangjie Xie ◽

Qi He ◽

Jie Li ◽

Jiali Liu ◽

...

Keyword(s):

Gene Expression ◽

Reference Genes ◽

Expression Patterns ◽

Regulatory Mechanisms ◽

Future Research ◽

Transcriptome Data ◽

Accurate Analysis ◽

Report Analysis ◽

The Stability ◽

Selection Of

Accurate analysis of gene expression requires selection of appropriate reference genes. In this study, we report analysis of eight candidate reference genes (ACTIN, UBQ, EF-1α, UBC, IF-4α, TUB, PP2A, and HIS), which were screened from the genome and transcriptome data in Brassica juncea. Four statistical analysis softwares geNorm, NormFinder, BestKeeper, and RefFinder were used to test the reliability and stability of gene expression of the reference genes. To further validate the stability of reference genes, the expression levels of two CYCD3 genes (BjuB045330 and BjuA003219) were studied. In addition, all genes in the xyloglucan endotransglucosylase/hydrolase (XTH) family were identified in B. juncea and their patterns at different periods of stem enlargement were analyzed. Results indicated that UBC and TUB genes showed stable levels of expression and are recommended for future research. In addition, XTH genes were involved in regulation of stem enlargement expression. These results provide new insights for future research aiming at exploring important functional genes, their expression patterns and regulatory mechanisms for mustard development.

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