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Open Access Highly Accessed Research article

RNA-seq: impact of RNA degradation on transcript quantification

Irene Gallego Romero1, Athma A Pai12, Jenny Tung13 and Yoav Gilad1*

Author Affiliations

1 Department of Human Genetics, University of Chicago, 920 E 58th St, CLSC 317, Chicago, IL 60637, USA

2 Present address: Department of Biology, Massachusetts Institute of Technology, 31 Ames Street, 68-271A, Cambridge, MA 02139-4307, USA

3 Present address: Department of Evolutionary Anthropology and Duke Population Research Institute, Duke University, 125 Science Drive, Durham, NC 27708, USA

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BMC Biology 2014, 12:42  doi:10.1186/1741-7007-12-42

Published: 30 May 2014

Abstract

Background

The use of low quality RNA samples in whole-genome gene expression profiling remains controversial. It is unclear if transcript degradation in low quality RNA samples occurs uniformly, in which case the effects of degradation can be corrected via data normalization, or whether different transcripts are degraded at different rates, potentially biasing measurements of expression levels. This concern has rendered the use of low quality RNA samples in whole-genome expression profiling problematic. Yet, low quality samples (for example, samples collected in the course of fieldwork) are at times the sole means of addressing specific questions.

Results

We sought to quantify the impact of variation in RNA quality on estimates of gene expression levels based on RNA-seq data. To do so, we collected expression data from tissue samples that were allowed to decay for varying amounts of time prior to RNA extraction. The RNA samples we collected spanned the entire range of RNA Integrity Number (RIN) values (a metric commonly used to assess RNA quality). We observed widespread effects of RNA quality on measurements of gene expression levels, as well as a slight but significant loss of library complexity in more degraded samples.

Conclusions

While standard normalizations failed to account for the effects of degradation, we found that by explicitly controlling for the effects of RIN using a linear model framework we can correct for the majority of these effects. We conclude that in instances in which RIN and the effect of interest are not associated, this approach can help recover biologically meaningful signals in data from degraded RNA samples.

Keywords:
RNA degradation; RIN; degradation; RNA; RNA-seq