A range finding protocol to support design for transcriptomics experimentation: examples of in-vitro and in-vivo murine UV exposure.

Oskar Bruning,Han Rauwerda,Mark De Jong,Timo M Breit,Martijs J Jonker,Annemieke De Vries,Wendy Rodenburg,Wim A Ensink,Conny T Van Oostrom,Rob J Dekker

doi:10.1371/journal.pone.0097089

Abstract

In transcriptomics research, design for experimentation by carefully considering biological, technological, practical and statistical aspects is very important, because the experimental design space is essentially limitless. Usually, the ranges of variable biological parameters of the design space are based on common practices and in turn on phenotypic endpoints. However, specific sub-cellular processes might only be partially reflected by phenotypic endpoints or outside the associated parameter range. Here, we provide a generic protocol for range finding in design for transcriptomics experimentation based on small-scale gene-expression experiments to help in the search for the right location in the design space by analyzing the activity of already known genes of relevant molecular mechanisms. Two examples illustrate the applicability: in-vitro UV-C exposure of mouse embryonic fibroblasts and in-vivo UV-B exposure of mouse skin. Our pragmatic approach is based on: framing a specific biological question and associated gene-set, performing a wide-ranged experiment without replication, eliminating potentially non-relevant genes, and determining the experimental ‘sweet spot’ by gene-set enrichment plus dose-response correlation analysis. Examination of many cellular processes that are related to UV response, such as DNA repair and cell-cycle arrest, revealed that basically each cellular (sub-) process is active at its own specific spot(s) in the experimental design space. Hence, the use of range finding, based on an affordable protocol like this, enables researchers to conveniently identify the ‘sweet spot’ for their cellular process of interest in an experimental design space and might have far-reaching implications for experimental standardization.

Highlights

Design for experimentation plays an important role in transcriptomics research
As no information was available to determine several of the in-vivo constant experimental parameters, we performed a number of tests that determined the optimal biopsy punch diameter size and RNA isolation protocol [12], as well as the maximum number of subsequent biopsy sampling from the same animal
This study addresses several issues that are involved with design for transcriptomics experimentation

Summary

Introduction

Design for experimentation plays an important role in transcriptomics research. There are several aspects that need to be considered: biological, technological, statistical and practical. The technological, e.g. choice of (microarray) platform, or practical, e.g. available budget, considerations are of great importance, but depend mostly on the individual experimenter’s setting This leaves biological aspects, such as those related to time, dose and space, which are frequently intuitively considered or based on common practice. In molecular biology based research, common practices in perturbation experiments are by tradition regularly tuned to phenotypic observations, such as the apoptosis, cellular responses, or cell growth. This could be based on the assumption that these measurable phenotypic endpoints coincide with changes in gene expression that are directly relevant to the mechanism under study, which might not always be the case. The selection of optimal experimental ranges within the design space should be an integral part of transcriptomics experimentation

Methods

Results

Conclusion