Internal Reference Gene Selection under Different Hormone Stresses in Multipurpose Timber Yielding Tree Neolamarckia cadamba

Neolamarckia cadamba, a member of the Rubiaceae family, is widely distributed throughout South Asia and South China. In order to acquire reliable and repeatable results, the use of a suitable internal reference gene to normalize the RT-qPCR data is essential. In this study, we reported the validation of housekeeping genes to identify the most suitable internal reference gene(s) for normalization of qPCR data obtained among different tissues (bud, leaf, cambium region) under different hormone stresses. Here, ΔCt, geNorm, NormFinder, and BestKeeper analyses were carried out to analyze the normalization of qPCR data of twenty-one reference gene families (ACT, CAC, CYP, EF1α, eIF, FPS1, FBK, GAPDH, RAN, PEPKR1, PP2A, RPL, RPS, RuBP, SAMDC, TEF, Tub-α, Tub-β, UBCE, UBQ, UPL) including 43 genes. The results showed that FPS1, RPL, and FBK were the most stable reference genes across all of the tested samples. In addition, the expression of NcEXPA8, one gene of interest that plays an important role in regulating cell wall extension, under different phytohormone stresses was used to further confirm the validated reference genes. Taken together, our results provide guidelines for reference gene selection under different phytohormone stresses and a foundation for more accurate and widespread use of RT-qPCR in N. cadamba.

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Internal Reference Gene Selection for Quantitative Real-Time RT-PCR Normalization in Potato Tissues

Phyton ◽

10.32604/phyton.2020.08874 ◽

2020 ◽

Vol 89 (2) ◽

pp. 329-344

Author(s):

Gang Li ◽

Yao Zhou ◽

Yaqi Zhao ◽

Yaxue Liu ◽

Yuwei Ke ◽

...

Keyword(s):

Real Time ◽

Reference Gene ◽

Gene Selection ◽

Rt Pcr ◽

Internal Reference ◽

Reference Gene Selection ◽

Internal Reference Gene ◽

Selection For

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Selection of internal references for RT-qPCR assays in Neurofibromatosis type 1 (NF1) related Schwann cell lines

PLoS ONE ◽

10.1371/journal.pone.0241821 ◽

2021 ◽

Vol 16 (2) ◽

pp. e0241821

Author(s):

Yi-Hui Gu ◽

Xi-Wei Cui ◽

Jie-Yi Ren ◽

Man-Mei Long ◽

Wei Wang ◽

...

Keyword(s):

Schwann Cell ◽

Cell Lines ◽

Reference Gene ◽

Reference Genes ◽

Gene Selection ◽

Neurofibromatosis Type ◽

Internal Reference ◽

Real Time Quantitative Pcr ◽

Internal Reference Gene

Real-time quantitative PCR (RT-qPCR) has been widely applied in uncovering disease mechanisms and screening potential biomarkers. Internal reference gene selection determines the accuracy and reproducibility of data analyses. The aim of this study was to identify the optimal reference genes for the relative quantitative analysis of RT-qPCR in fourteen NF1 related cell lines, including non-tumor, benign and malignant Schwann cell lines. The expression characteristics of eleven candidate reference genes (RPS18, ACTB, B2M, GAPDH, PPIA, HPRT1, TBP, UBC, RPLP0, TFRC and RPL32) were screened and analyzed by four software programs: geNorm, NormFinder, BestKeeper and RefFinder. Results showed that GAPDH, the most frequently used internal reference gene, was significantly unstable between various cell lines. The combinational use of two reference genes (PPIA and TBP) was optimal in malignant Schwann cell lines and the use of single reference genes (PPIA or PRLP0) alone or in combination was optimal in benign Schwann cell lines. These recommended internal reference gene selections may improve the accuracy and reproducibility of RT-qPCR in gene expression analyses of NF1 related tumors.

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Current Practices for Reference Gene Selection in RT-qPCR of Aspergillus: Outlook and Recommendations for the Future

Genes ◽

10.3390/genes12070960 ◽

2021 ◽

Vol 12 (7) ◽

pp. 960

Author(s):

Meagan Archer ◽

Jianping Xu

Keyword(s):

Gene Expression ◽

Reference Gene ◽

Reference Genes ◽

Gene Selection ◽

Quantitative Polymerase Chain Reaction ◽

Specific Method ◽

Experimental Conditions ◽

Reference Gene Selection ◽

Physiological Processes ◽

The Stability

Aspergillus is a genus of filamentous fungi with vast geographic and ecological distributions. Species within this genus are clinically, agriculturally and biotechnologically relevant, leading to increasing interest in elucidating gene expression dynamics of key metabolic and physiological processes. Reverse-transcription quantitative Polymerase Chain Reaction (RT-qPCR) is a sensitive and specific method of quantifying gene expression. A crucial step for comparing RT-qPCR results between strains and experimental conditions is normalisation to experimentally validated reference gene(s). In this review, we provide a critical analysis of current reference gene selection and validation practices for RT-qPCR gene expression analyses of Aspergillus. Of 90 primary research articles obtained through our PubMed query, 17 experimentally validated the reference gene(s) used. Twenty reference genes were used across the 90 studies, with beta-tubulin being the most used reference gene, followed by actin, 18S rRNA and glyceraldehyde 3-phosphate dehydrogenase. Sixteen of the 90 studies used multiple reference genes for normalisation. Failing to experimentally validate the stability of reference genes can lead to conflicting results, as was the case for four studies. Overall, our review highlights the need to experimentally validate reference genes in RT-qPCR studies of Aspergillus.

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Reference gene selection and evaluation for expression analysis using qRT-PCR in Galeruca daurica (Joannis)

Bulletin of Entomological Research ◽

10.1017/s0007485316000948 ◽

2016 ◽

Vol 107 (3) ◽

pp. 359-368 ◽

Cited By ~ 12

Author(s):

Y. Tan ◽

X.-R. Zhou ◽

B.-P. Pang

Keyword(s):

Reference Gene ◽

Reference Genes ◽

Target Genes ◽

Gene Selection ◽

Developmental Stages ◽

Pcr Analysis ◽

Experimental Conditions ◽

Reference Gene Selection ◽

Functional Studies ◽

Qrt Pcr

AbstractQuantitative real-time PCR (qRT-PCR) has been used extensively to analyze gene expression and decipher gene function. To obtain the optimal and stable normalization factors for qRT-PCR, selection and validation of reference genes should be conducted in diverse conditions. In insects, more and more studies confirmed the necessity and importance of reference gene selection. In this study, eight traditionally used reference genes in Galeruca daurica (Joannis) were assessed, using qRT-PCR, for suitability as normalization genes under different experimental conditions using four statistical programs: geNorm, Normfinder, BestKeeper and the comparative ΔCt method. The genes were ranked from the most stable to the least stable using RefFinder. The optimal suite of recommended reference genes was as follows: succinate dehydrogenase (SDHA) and tubulin-alpha (TUB-α) for temperature-treated larvae; ribosomal protein L32, SDHA and glutathione S-transferase were best for all developmental stages; ACT and TUB-α for male and female adults; SDHA and TUB-α were relatively stable and expressed in different tissues, both diapause and non-diapause adults. Reference gene evaluation was validated using expression of two target genes: the P450 CYP6 gene and the heat shock protein gene Hsp70. These results confirm the importance of custom reference gene selection when studies are conducted under diverse experimental conditions. A standardized qRT-PCR analysis procedure for gene functional studies is provided that could be useful in studies on other insect species.

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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.

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Reference Gene Selection for Normalizing Gene Expression in Ips Sexdentatus (Coleoptera: Curculionidae: Scolytinae) Under Different Experimental Conditions

Frontiers in Physiology ◽

10.3389/fphys.2021.752768 ◽

2021 ◽

Vol 12 ◽

Author(s):

Gothandapani Sellamuthu ◽

Shan Amin ◽

Jan Bílý ◽

Jirí Synek ◽

Roman Modlinger ◽

...

Keyword(s):

Gene Expression ◽

Reference Gene ◽

Expression Analysis ◽

Reference Genes ◽

Gene Expression Analysis ◽

Gene Selection ◽

Experimental Conditions ◽

Tissue Specific ◽

Reference Gene Selection ◽

Ips Sexdentatus

Ips sexdentatus (Coleoptera: Curculionidae: Scolytinae) is one of the most destructive and economically important forest pests. A better understanding of molecular mechanisms underlying its adaptation to toxic host compounds may unleash the potential for future management of this pest. Gene expression studies could be considered as one of the key experimental approaches for such purposes. A suitable reference gene selection is fundamental for quantitative gene expression analysis and functional genomics studies in I. sexdentatus. Twelve commonly used reference genes in Coleopterans were screened under different experimental conditions to obtain accurate and reliable normalization of gene expression data. The majority of the 12 reference genes showed a relatively stable expression pattern among developmental stages, tissue-specific, and sex-specific stages; however, some variabilities were observed during varied temperature incubation. Under developmental conditions, the Tubulin beta-1 chain (β-Tubulin) was the most stable reference gene, followed by translation elongation factor (eEF2) and ribosomal protein S3 (RPS3). In sex-specific conditions, RPS3, β-Tubulin, and eEF2 were the most stable reference genes. In contrast, different sets of genes were shown higher stability in terms of expression under tissue-specific conditions, i.e., RPS3 and eEF2 in head tissue, V-ATPase-A and eEF2 in the fat body, V-ATPase-A and eEF2 in the gut. Under varied temperatures, β-Tubulin and V-ATPase-A were most stable, whereas ubiquitin (UbiQ) and V-ATPase-A displayed the highest expression stability after Juvenile Hormone III treatment. The findings were validated further using real-time quantitative reverse transcription PCR (RT-qPCR)-based target gene expression analysis. Nevertheless, the present study delivers a catalog of reference genes under varied experimental conditions for the coleopteran forest pest I. sexdentatus and paves the way for future gene expression and functional genomic studies on this species.

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A simple method to assess group difference in RT-qPCR reference gene selection using GeNorm: The case of the placental sex

Scientific Reports ◽

10.1038/s41598-017-16916-y ◽

2017 ◽

Vol 7 (1) ◽

Cited By ~ 12

Author(s):

Joey St-Pierre ◽

Jean-Charles Grégoire ◽

Cathy Vaillancourt

Keyword(s):

Reference Gene ◽

Gene Selection ◽

Simple Method ◽

Reference Gene Selection

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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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