Gene expression analysis of skin grafts and cultured keratinocytes using synthetic RNA normalization reveals insights into differentiation and growth control

Shintaro Katayama,Tiina Skoog,Anna Johnsson,Eeva-Mari Jouhilahti,Outi Elomaa,Sten Linnarsson,Esko Kankuri,Jyrki Vuola,Juha Kere,Mari H Tervaniemi,Kristo Nuutila,H Annika Siitonen,Peter Lönnerberg

doi:10.1186/s12864-015-1671-5

Abstract

BackgroundKeratinocytes (KCs) are the most frequent cells in the epidermis, and they are often isolated and cultured in vitro to study the molecular biology of the skin. Cultured primary cells and various immortalized cells have been frequently used as skin models but their comparability to intact skin has been questioned. Moreover, when analyzing KC transcriptomes, fluctuation of polyA+ RNA content during the KCs’ lifecycle has been omitted.ResultsWe performed STRT RNA sequencing on 10 ng samples of total RNA from three different sample types: i) epidermal tissue (split-thickness skin grafts), ii) cultured primary KCs, and iii) HaCaT cell line. We observed significant variation in cellular polyA+ RNA content between tissue and cell culture samples of KCs. The use of synthetic RNAs and SAMstrt in normalization enabled comparison of gene expression levels in the highly heterogenous samples and facilitated discovery of differences between the tissue samples and cultured cells. The transcriptome analysis sensitively revealed genes involved in KC differentiation in skin grafts and cell cycle regulation related genes in cultured KCs and emphasized the fluctuation of transcription factors and non-coding RNAs associated to sample types.ConclusionsThe epidermal keratinocytes derived from tissue and cell culture samples showed highly different polyA+ RNA contents. The use of SAMstrt and synthetic RNA based normalization allowed the comparison between tissue and cell culture samples and thus proved to be valuable tools for RNA-seq analysis with translational approach. Transciptomics revealed clear difference both between tissue and cell culture samples and between primary KCs and immortalized HaCaT cells.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-015-1671-5) contains supplementary material, which is available to authorized users.

Highlights

Keratinocytes (KCs) are the most frequent cells in the epidermis, and they are often isolated and cultured in vitro to study the molecular biology of the skin
The Split-thickness skin graft (SG) samples were used directly for total RNA extraction whereas the full-thickness biopsies were used to set up KC cultures from which total RNA was extracted at early (1st; Cultured keratinocyte at early passage (EKC)) and late (5th-6th; Cultured keratinocyte at late passage (LKC)) passages
We found that the estimated polyA+ RNA contents as quantified against the added spike-in RNA controls varied in different sample types (Additional file 5: Figure S1)

Summary

Results

We performed STRT RNA sequencing on 10 ng samples of total RNA from three different sample types: i) epidermal tissue (split-thickness skin grafts), ii) cultured primary KCs, and iii) HaCaT cell line. We observed significant variation in cellular polyA+ RNA content between tissue and cell culture samples of KCs. The use of synthetic RNAs and SAMstrt in normalization enabled comparison of gene expression levels in the highly heterogenous samples and facilitated discovery of differences between the tissue samples and cultured cells. The transcriptome analysis sensitively revealed genes involved in KC differentiation in skin grafts and cell cycle regulation related genes in cultured KCs and emphasized the fluctuation of transcription factors and non-coding RNAs associated to sample types

Conclusions

Background

Results and discussion

Conclusion

Methods