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Open Access Highly Accessed Research article

Impact of RNA degradation on gene expression profiling

Lennart Opitz1, Gabriela Salinas-Riester1, Marian Grade2, Klaus Jung3, Peter Jo2, Georg Emons2, B Michael Ghadimi2, Tim Beißbarth3* and Jochen Gaedcke2

Author Affiliations

1 DNA Microarray Facility, University Medicine Göttingen, Humboldtallee 23, 37073 Göttingen, Germany

2 Department Surgery, University Medicine Göttingen, Robert-Koch-Str. 40, 37073 Göttingen, Germany

3 Department Medical Statistics, University Medicine Göttingen, Humboldtallee 32, 37073 Göttingen, Germany

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BMC Medical Genomics 2010, 3:36  doi:10.1186/1755-8794-3-36

Published: 9 August 2010

Abstract

Background

Gene expression profiling is a highly sensitive technique which is used for profiling tumor samples for medical prognosis. RNA quality and degradation influence the analysis results of gene expression profiles. The impact of this influence on the profiles and its medical impact is not fully understood. As patient samples are very valuable for clinical studies, it is necessary to establish criteria for the RNA quality to be able to use these samples in later analysis.

Methods

To investigate the effects of RNA integrity on gene expression profiling, whole genome expression arrays were used. We used tumor biopsies from patients diagnosed with locally advanced rectal cancer. To simulate degradation, the isolated total RNA of all patients was subjected to heat-induced degradation in a time-dependent manner. Expression profiling was then performed and data were analyzed bioinformatically to assess the differences.

Results

The differences introduced by RNA degradation were largely outweighed by the biological differences between the patients. Only a relatively small number of probes (275 out of 41,000) show a significant effect due to degradation. The genes that show the strongest effect due to RNA degradation were, especially, those with short mRNAs and probe positions near the 5' end.

Conclusions

Degraded RNA from tumor samples (RIN > 5) can still be used to perform gene expression analysis. A much higher biological variance between patients is observed compared to the effect that is imposed by degradation of RNA. Nevertheless there are genes, very short ones and those with the probe binding side close to the 5' end that should be excluded from gene expression analysis when working with degraded RNA. These results are limited to the Agilent 44 k microarray platform and should be carefully interpreted when transferring to other settings.