Email updates

Keep up to date with the latest news and content from BMC Clinical Pathology and BioMed Central.

Open Access Research article

Transcriptional profiling of degraded RNA in cryopreserved and fixed tissue samples obtained at autopsy

Andrew C Haller1, Deepa Kanakapalli1, Rosemarie Walter2, Samir Alhasan2, James F Eliason2 and Richard B Everson13*

Author Affiliations

1 Wayne State University, Barbara Ann Karmanos Cancer Institute, Detroit, MI, USA

2 Asterand plc., Detroit, MI, USA

3 Wayne State University School of Medicine, Departments of Medicine and Pathology, Detroit, MI, USA

For all author emails, please log on.

BMC Clinical Pathology 2006, 6:9  doi:10.1186/1472-6890-6-9

Published: 4 December 2006

Abstract

Background

Traditional multiplexed gene expression methods require well preserved, intact RNA. Such specimens are difficult to acquire in clinical practice where formalin fixation is the standard procedure for processing tissue. Even when special handling methods are used to obtain frozen tissue, there may be RNA degradation; for example autopsy samples where degradation occurs both pre-mortem and during the interval between death and cryopreservation. Although specimens with partially degraded RNA can be analyzed by qRT-PCR, these analyses can only be done individually or at low levels of multiplexing and are laborious and expensive to run for large numbers of RNA targets.

Methods

We evaluated the ability of the cDNA-mediated Annealing, Selection, extension, and Ligation (DASL) assay to provide highly multiplexed analyses of cryopreserved and formalin fixed, paraffin embedded (FFPE) tissues obtained at autopsy. Each assay provides data on 1536 targets, and can be performed on specimens with RNA fragments as small as 60 bp.

Results

The DASL performed accurately and consistently with cryopreserved RNA obtained at autopsy as well as with RNA extracted from formalin-fixed paraffin embedded tissue that had a cryopreserved mirror image specimen with high quality RNA. In FFPE tissue where the cryopreserved mirror image specimen was of low quality the assay performed reproducibly on some but not all specimens.

Conclusion

The DASL assay provides reproducible results from cryopreserved specimens and many FFPE specimens obtained at autopsy. Gene expression analyses of these specimens may be especially valuable for the study of non-cancer endpoints, where surgical specimens are rarely available.