Cancer Diagnosis Program | Biorepositories and Biospecimen Research Branch

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RNA extraction for RNA sequencing of archival renal tissues.

Author(s): Landolt L, Marti HP, Beisland C, Flatberg A, Eikrem OS

Publication: Scand J Clin Lab Invest, 2016, Vol. , Page 1-9

PubMed ID: 27173776 PubMed Review Paper? No

Purpose of Paper

Conclusion of Paper

Studies

1. Study Purpose

   This study compared the yield, integrity, and purity of RNA extracted from whole FFPE sections and microdissected glomeruli using four different kits and determined the suitability of the extracted RNA for next-generation sequencing.  Fresh 10 µm sections of FFPE normal adjacent tissue obtained from 16-gauge biopsies from two patients with clear cell renal carcinoma were immediately placed in tubes for deparaffinization in xylene. Microdissected glomeruli or whole sections were placed in lysis buffer for extraction with the High Pure kit, PKD buffer for extraction with the miRNeasy or RNeasy FFPE kits, or water for extraction with the ExpressArt kit and RNA was extracted. RNA was quantified using NanoDrop and Qubit, integrity was verified by DV200 on a Bioanalyzer, and NGS sequencing was performed using the TruSeq RNA Access Library Preparation Kit.

   Summary of Findings:

   Average RNA yields from whole sections as measured by NanoDrop and Qubit were highest using the HighPure kit (137 ng and 102 ng, respectively) followed by the ExpressArt kit (84 ng and 92 ng, respectively) with the lowest yields obtained when extraction was with the miRNeasy (53 ng and 54 ng, respectively) or RNeasy (64 ng and 62 ng, respectively) kits. When RNA was extracted from 80-160 microdissected glomeruli the yield as measured by NanoDrop was highest when extraction was performed with the ExpressArt kit (138 ng) followed by the RNeasy kit (114 ng), the HighPure kit (54 ng), and the miRNeasy kit (32 ng), but the yield from each kits was too low for quantification by Qubit. The mean 260/280 ratios of RNA extracted from sections or microdissected glomeruli were also highest when extraction was performed with the HighPure kit (1.8 and 1.5, respectively), ExpressArt (1.9 and 1.5, respectively), or RNeasy (1.9 and 1.3, respectively) kits with lower ratios observed for RNA extracted with the miRNeasy kit (1.5 and 1.1, respectively).  The DV200 values from whole sections and microdissected glomeruli were highest when extraction was with the HighPure (76% and 89%, respectively) or ExpressArt (72% and 82%, respectively) kits and lower when extracted with the miRNeasy (51% and 26%, respectively) or RNeasy (62% and 52%, respectively) kits. The highest average number of reads and highest number of reads mapped to the genome or transcriptome was generated when RNA was extracted using the HighPure kit (24.1 M and 20.8 M, respectively) followed by the miRNAeasy (12.3 M and 10.1 M, respectively) and ExpressArt kits (10.6 M and 9.1 M, respectively). 

   Biospecimens

   • Tissue - Kidney

   Preservative Types

   • Formalin

   Diagnoses:

   • Neoplastic - Normal Adjacent

   Platform:

   Analyte	Technology Platform
   RNA	Automated electrophoresis/Bioanalyzer
   RNA	Next generation sequencing
   RNA	Spectrophotometry
   RNA	Fluorometry

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Biospecimen Aliquots and Components	Aliquot size/volume	Whole section of 16-G biopsy 80-160 microdissected glomeruli
   Spectrophotometry Specific	Technology platform	Qubit Bioanalyzer
   Analyte Extraction and Purification	Analyte isolation method	miRNeasy FFPE kit RNeasy FFPE kit ExpressArt Clear FFPE RNAready kit High Pure FFPE RNA Isolation kit

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