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Validation of RNA Extraction Methods and Suitable Reference Genes for Gene Expression Studies in Developing Fetal Human Inner Ear Tissue

Lookup NU author(s): Jasmin TurnerORCiD



This work is licensed under a Creative Commons Attribution 4.0 International License (CC BY 4.0).


© 2024 by the authors.A comprehensive gene expression investigation requires high-quality RNA extraction, in sufficient amounts for real-time quantitative polymerase chain reaction and next-generation sequencing. In this work, we compared different RNA extraction methods and evaluated different reference genes for gene expression studies in the fetal human inner ear. We compared the RNA extracted from formalin-fixed paraffin-embedded tissue with fresh tissue stored at −80 °C in RNAlater solution and validated the expression stability of 12 reference genes (from gestational week 11 to 19). The RNA from fresh tissue in RNAlater resulted in higher amounts and a better quality of RNA than that from the paraffin-embedded tissue. The reference gene evaluation exhibited four stably expressed reference genes (B2M, HPRT1, GAPDH and GUSB). The selected reference genes were then used to examine the effect on the expression outcome of target genes (OTOF and TECTA), which are known to be regulated during inner ear development. The selected reference genes displayed no differences in the expression profile of OTOF and TECTA, which was confirmed by immunostaining. The results underline the importance of the choice of the RNA extraction method and reference genes used in gene expression studies.

Publication metadata

Author(s): Steinacher C, Rieder D, Turner JE, Solanky N, Nishio S-Y, Usami S-I, Hausott B, Schrott-Fischer A, Dudas J

Publication type: Article

Publication status: Published

Journal: International Journal of Molecular Sciences

Year: 2024

Volume: 25

Issue: 5

Online publication date: 02/03/2024

Acceptance date: 28/02/2024

Date deposited: 25/03/2024

ISSN (print): 1661-6596

ISSN (electronic): 1422-0067

Publisher: MDPI


DOI: 10.3390/ijms25052907

PubMed id: 38474154


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