Abstract
Melanoma is characterized by a high mutation rate, and selection of the proper reference for RT-qPCR 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; this affects the interpretation of the results and the drawing of conclusions. For reliable assessment of changes in B4GALT gene expression in melanoma, and for comparison with their expression levels in melanocytes, we implemented our innovative GenExpA software, selecting 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 an absolutely 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. GenExpA works in a manner independent of the experimental model and the normalizer. GenExpA is available at https://github.com/DorotaHojaLukowicz/GenExpA or https://www.sciencemarket.pl/baza-programow-open-source#oferty.
Highlights GenExpA – next-generation software for normalizer selection and validation
The GenExpA tool defines how low the stability value of the reference should be
The GenExpA tool determines the level of gene expression analysis robustness
The GenExpA tool increases the speed of analysis and reduces cost
Competing Interest Statement
The authors have declared no competing interest.
Footnotes
e-mail addresses: dorota.hoja-lukowicz{at}uj.edu.pl, dawid.maciazek{at}gmail.com, marcelina.janik{at}uj.edu.pl
https://www.sciencemarket.pl/baza-programow-open-source#oferty