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Valuable lessons-learned in transcriptomics experimentation.

Bruning O, Rauwerda H, Dekker RJ, de Leeuw WC, Wackers PF, Ensink WA, Jonker MJ, Breit TM - Transcription (2015)

Bottom Line: We have collected several valuable lessons that will help improve transcriptomics experimentation.These lessons relate to experiment design, execution, and analysis.The cautions, but also the pointers, may help biologists avoid common pitfalls in transcriptomics experimentation and achieve better results with their transcriptome studies.

View Article: PubMed Central - PubMed

Affiliation: a RNA Biology & Applied Bioinformatics research group; Swammerdam Institute for Life Sciences; Faculty of Science; University of Amsterdam ; Amsterdam , the Netherlands.

ABSTRACT
We have collected several valuable lessons that will help improve transcriptomics experimentation. These lessons relate to experiment design, execution, and analysis. The cautions, but also the pointers, may help biologists avoid common pitfalls in transcriptomics experimentation and achieve better results with their transcriptome studies.

Show MeSH
Smoothing effects of improper averaging over individuals. Time profiles of Log2 fold change compared to t = 0 of the Mdm2 gene in skin derived from an in vitro mouse study. The mice were treated with UV-B at t = 0. (A) Averaged profiles over biological replicates for both treated and untreated samples. (B) Profiles of individual mice that were not treated. Different colors indicate individual mice. (C) Profiles of individual mice that were irradiated with a high dose of UV-B. Different colors indicate individual mice.
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f0001: Smoothing effects of improper averaging over individuals. Time profiles of Log2 fold change compared to t = 0 of the Mdm2 gene in skin derived from an in vitro mouse study. The mice were treated with UV-B at t = 0. (A) Averaged profiles over biological replicates for both treated and untreated samples. (B) Profiles of individual mice that were not treated. Different colors indicate individual mice. (C) Profiles of individual mice that were irradiated with a high dose of UV-B. Different colors indicate individual mice.

Mentions: Unfortunately, range finding is not always sufficient. In vivo studies also need to be tested for inter-individual differences. If these are too diverse on a basal transcriptome level for the tissue under study, they cannot be considered replicates anymore. This effectively inhibits standard transcriptomics experimentation and a different experimental approach should be found. Examples of how improper replicates can be deceiving are our encounters, on several occasions, with very smooth looking profiles of average gene expression, suggesting a relation between the observed experimental factor and gene expression, whereas looking at the profiles from the underlying individual animals, no rhyme or reason could be detected (Fig. 1).Figure 1.


Valuable lessons-learned in transcriptomics experimentation.

Bruning O, Rauwerda H, Dekker RJ, de Leeuw WC, Wackers PF, Ensink WA, Jonker MJ, Breit TM - Transcription (2015)

Smoothing effects of improper averaging over individuals. Time profiles of Log2 fold change compared to t = 0 of the Mdm2 gene in skin derived from an in vitro mouse study. The mice were treated with UV-B at t = 0. (A) Averaged profiles over biological replicates for both treated and untreated samples. (B) Profiles of individual mice that were not treated. Different colors indicate individual mice. (C) Profiles of individual mice that were irradiated with a high dose of UV-B. Different colors indicate individual mice.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4581358&req=5

f0001: Smoothing effects of improper averaging over individuals. Time profiles of Log2 fold change compared to t = 0 of the Mdm2 gene in skin derived from an in vitro mouse study. The mice were treated with UV-B at t = 0. (A) Averaged profiles over biological replicates for both treated and untreated samples. (B) Profiles of individual mice that were not treated. Different colors indicate individual mice. (C) Profiles of individual mice that were irradiated with a high dose of UV-B. Different colors indicate individual mice.
Mentions: Unfortunately, range finding is not always sufficient. In vivo studies also need to be tested for inter-individual differences. If these are too diverse on a basal transcriptome level for the tissue under study, they cannot be considered replicates anymore. This effectively inhibits standard transcriptomics experimentation and a different experimental approach should be found. Examples of how improper replicates can be deceiving are our encounters, on several occasions, with very smooth looking profiles of average gene expression, suggesting a relation between the observed experimental factor and gene expression, whereas looking at the profiles from the underlying individual animals, no rhyme or reason could be detected (Fig. 1).Figure 1.

Bottom Line: We have collected several valuable lessons that will help improve transcriptomics experimentation.These lessons relate to experiment design, execution, and analysis.The cautions, but also the pointers, may help biologists avoid common pitfalls in transcriptomics experimentation and achieve better results with their transcriptome studies.

View Article: PubMed Central - PubMed

Affiliation: a RNA Biology & Applied Bioinformatics research group; Swammerdam Institute for Life Sciences; Faculty of Science; University of Amsterdam ; Amsterdam , the Netherlands.

ABSTRACT
We have collected several valuable lessons that will help improve transcriptomics experimentation. These lessons relate to experiment design, execution, and analysis. The cautions, but also the pointers, may help biologists avoid common pitfalls in transcriptomics experimentation and achieve better results with their transcriptome studies.

Show MeSH