RT-qPCR analysis of human melanoma progression-related genes – A novel workflow for selection and validation of candidate reference genes

Janik, Marcelina; Szwed, Sabina; Grzmil, Paweł; Kaczmarek, Radosław; Czerwiński, Marcin; Hoja-Łukowicz, Dorota

doi:10.1016/j.biocel.2018.05.007

Cited by 6 publications

(15 citation statements)

References 22 publications

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“…Human melanoma cell line WM35 (cutaneous primary, radial/vertical growth phase) was kindly donated by Prof. A. Mackiewicz of the Department of Cancer Immunology, University School of Medical Sciences, Greater Poland Cancer Center, Poznań, Poland. Cell lines were cultured as described previously [12]. All cultures were verified mycoplasma-free using the MycoAlert mycoplasma detection kit (Lonza).…”

Section: Methodsmentioning

confidence: 99%

“…Human epidermal melanocytes (adult, HEMa-LP) and human melanoma cell lines Mel202 (uveal primary), WM35 (cutaneous primary, radial/vertical growth phase), WM793 (cutaneous primary, vertical growth phase) and WM266-4 (skin metastasis) were cultured as described previously (12). All cultures were verified mycoplasma-free using the MycoAlert™ mycoplasma detection kit (Lonza).…”

Section: Cell Culturementioning

confidence: 99%

“…Total RNA extraction, the reverse-transcription reaction and real-time qPCR reactions were performed as previously described (12). RNA and cDNA concentrations as well as their quality were assessed with a NanoDrop 2000 spectrophotometer (Supplementary Data 1).…”

Section: Reverse Transcription Qpcrmentioning

confidence: 99%

“…We demonstrated that beyond the parent group of samples of interest (experimental model) it is necessary to construct daughter groups (auxiliary modelscombinations of samples without repetition), followed by selection and validation of an appropriate normalizer for each group. If we get inconsistent results for the expression level of a target gene based on a comparison of individual samples through all groups, we have to search for new references with improved stability values via progressive removal of the least stable candidate reference gene in each group, followed by re-selection and re-validation of new normalizers each time (12). However, this proposed approach of RT-qPCR data analysis has been a very timeconsuming task, requiring the use of several specialist software packages.…”

Section: Introductionmentioning

confidence: 99%

“…Typically, one or more of five algorithms (geNorm [3], NormFinder [9], BestKeeper [10], the comparative ΔCt method [11] and RefFinder; http://150.216.56.64/referencegene.php) are used to select the best stable single or combination of reference genes from a panel of candidate genes, although there are also other proposed algorithms [12] The normalizer with the lowest stability value is considered the best and is then used for calculation of target gene expression in a group of samples. We have recently shown that this commonly accepted approach seems not enough for accurate and reliable target gene transcript normalization and that it may lead to biologically incorrect conclusions [13]. We demonstrated that beyond the parent model of samples of interest (experimental model), it is necessary to construct daughter models (auxiliary models built from the same samples as the experimental model but with fewer samplescombinations of samples of interest without repetition), followed by selection and validation of an appropriate normalizer for each model.…”

Section: Introductionmentioning

confidence: 99%

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Innovative GenExpA software for selecting suitable reference genes for reliable normalization of gene expression in melanoma

Hoja-Łukowicz

Maciążek

Janik

2021

Preprint

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Melanoma is characterized by a high mutation rate and the proper reference selection is a serious problem. The algorithms commonly used to select the best stable reference gene or pair of genes in RT-qPCR data analysis have their limitations which affects the interpretation of the results and drawing conclusions. For reliable assessment of the B4GALT genes expression changes in melanoma and comparing them to their expression levels in melanocytes, we implemented an innovative GenExpA software. We proposed selection of the best reference by combining the NormFinder algorithm with progressive removal of the least stable gene from the candidate genes in a given experimental model and the set of daughter models assigned to it. The reliability of references is validated by normalizing of target gene expression level through all models and is described by the coherence score. The software performs statistical analyses and generates results in the form of ready-to-use graphs and tables. GenExpA is a promising tool for basic research; it produces analyses of target gene expression in a manner independent of the experimental model and the normalizer. GenExpA and its manual is freely available at https://drive.google.com/drive/folders/1FcpBtEc_bKPvEnM5GcPZerBb_Tw_fCkt?usp=sharing or https://www.sciencemarket.pl/baza-programow-open-source#oferty. Supplemental materials are deposited under DOI number: 10.5281/zenodo.4544296.

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Section: Methodsmentioning

confidence: 99%

Section: Cell Culturementioning

confidence: 99%

Section: Reverse Transcription Qpcrmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 3 more Smart Citations

Innovative GenExpA software for selecting suitable reference genes for reliable normalization of gene expression in melanoma

Hoja-Łukowicz

Maciążek

Janik

2021

Preprint

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RETRACTED: Long non-coding RNA UCA1 targets miR-185-5p and regulates cell mobility by affecting epithelial-mesenchymal transition in melanoma via Wnt/β-catenin signaling pathway

Chen¹,

Gao²,

Yu³

et al. 2018

Gene

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Innovative GenExpA software for selecting suitable reference genes for reliable normalization of gene expression in melanoma

Hoja-Łukowicz

Maciążek

Kościelniak

et al. 2022

Sci Rep

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The algorithms commonly used to select the best stable reference gene in RT-qPCR data analysis have their limitations. We showed that simple selection of the reference gene or pair of genes with the lowest stability value from the pool of potential reference genes—a commonly used approach—is not sufficient to accurately and reliably normalize the target gene transcript and can lead to biologically incorrect conclusions. For reliable assessment of changes in a target gene expression level, we propose our innovative GenExpA software, which works in a manner independent of the experimental model and the normalizer used. GenExpA software selects the best reference by combining the NormFinder algorithm with progressive removal of the least stable gene from the candidate genes in a given experimental model and in the set of daughter models assigned to it. The reliability of references is validated based on the consistency of the statistical analyses of normalized target gene expression levels through all models, described by the coherence score (CS). The use of the CS value imparts a new quality to qPCR analysis because it clarifies how low the stability value of reference must be in order for biologically correct conclusions to be drawn. We tested our method on qPCR data for the B4GALT genes family in melanoma, which is characterized by a high mutation rate, and in melanocytes. GenExpA is available at https://github.com/DorotaHojaLukowicz/GenExpA or https://www.sciencemarket.pl/baza-programow-open-source#oferty.

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RT-qPCR analysis of human melanoma progression-related genes – A novel workflow for selection and validation of candidate reference genes

Cited by 6 publications

References 22 publications

Innovative GenExpA software for selecting suitable reference genes for reliable normalization of gene expression in melanoma

Innovative GenExpA software for selecting suitable reference genes for reliable normalization of gene expression in melanoma

RETRACTED: Long non-coding RNA UCA1 targets miR-185-5p and regulates cell mobility by affecting epithelial-mesenchymal transition in melanoma via Wnt/β-catenin signaling pathway

Innovative GenExpA software for selecting suitable reference genes for reliable normalization of gene expression in melanoma

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