RNA extraction for RNA sequencing of archival renal tissues

Abstract

Background: Next generation sequencing (NGS) and especially ribonucleic acid (RNA) sequencing is a powerful tool to acquire insights into molecular disease mechanisms. Therefore, it is of interest to optimize methods for RNA extraction from archival, formalin fixed and paraffin embedded (FFPE) tissues. This is challenging due to RNA degradation and chemical modifications. The aim of this study was to find the most appropriate method to extract RNA from FFPE renal tissue to enable NGS.

Method: We evaluated seven commercially available RNA extraction kits: High Pure FFPE RNA Isolation (Roche), ExpressArt Clear FFPE RNAready (Amsbio), miRNeasy FFPE, RNeasy FFPE (Qiagen), PureLink FFPE Total RNA (Invitrogen), RecoverAll Total Nucleic Acid Isolation (Ambion) and Absolutely RNA FFPE Kit (Agilent). RNA was obtained from tissue blocks of two healthy, male Wistar rats and from normal renal tissue of patients undergoing nephrectomy. Yield and quality of RNA extracted from rat whole kidney sections, human kidney core biopsies and laser capture microdissected (LCM) glomerular cross-sections were assessed: Analyses of RNA quantity were performed using NanoDrop and Qubit. RNA quality is reflected by DV200 values (% of RNA fragments >200 nucleotides) utilizing the Agilent 2100 BioAnalyzer. RNA of human LCM samples was subsequently sequenced using the Illumina TruSeq^® RNA Access Library Preparation Kit.

Conclusion: Total RNA can be extracted from archival renal biopsies in sufficient quality and quantity from one human kidney biopsy section and from around 100 LCM glomerular cross-sections to enable successful RNA library preparation and sequencing using commercially available RNA extraction kits.

Keywords:

• Formalin-fixed and paraffin embedded
• kidney biopsies
• laser capture microdissection
• next generation sequencing
• RNA extraction

Acknowledgements

We thank the local urologists for participation in biopsy harvesting. We thank the staff from the Genomics Core Facility (GCF), Norwegian University of Science and Technology (NTNU) for RNA library prep, sequencing and bioinformatics analysis. GCF is funded by the Faculty of Medicine at NTNU and the Central Norway Regional Health Authority.

Disclosure statement

The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.