Expression analysis on archival material revisited: isolation and quantification of RNA extracted from FFPE samples.
BACKGROUND
Formalin-fixed paraffin-embedded (FFPE) tissue is the most readily available source of RNA for the gene expression studies. The main disadvantage is the poor quality of isolated RNA. Our group recently compared 5 commercially available RNA isolation kits and concluded that the RNeasy FFPE kit from Qiagen was the most appropriate one. However, this kit has been discontinued and replaced by a new version. In this study both kits were compared, and spectrophotometric and fluorometric analyses for quantification of RNA samples extracted from FFPE tissue.


METHODS
Both RNeasy FFPE kits were compared for the total RNA and DNA yields, purity, and raw cycle threshold. Quantity and quality of the isolated RNA was measured using the NanoDrop ND-1000 spectrophotometer and Qubit 2.0 fluorometer.


RESULTS
The average concentration of RNA extracted from FFPE tissue measured using the NanoDrop was 32.0%±9.5% higher than the concentration measured using the Qubit. When measuring an RNA sample extracted from a cell line, the concentration measured using both methods was similar. When comparing both RNeasy FFPE kits, marginal differences were observed for total RNA yield, purity, and raw cycle threshold. However, the residual DNA in the samples isolated using the old kit was higher than in the samples isolated using the new kit.


CONCLUSIONS
A fluorometric analysis is more suitable for quantification of RNA samples extracted from FFPE tissue compared with spectrophotometric analysis. For RNA isolation from FFPE tissue, both old and new RNeasy FFPE kits were adequate. The new kit resulted in more efficient DNA removal.
CORE-Antwerp, Laboratory for Cancer Research and Clinical Oncology, University of Antwerp, Antwerp, Belgium. christophe.deben@ua.ac.be
Christophe
C
Deben
Karen
K
Zwaenepoel
Carolien
C
Boeckx
An
A
Wouters
Patrick
P
Pauwels
Marc
M
Peeters
Filip
F
Lardon
Marc
M
Baay
Vanessa
V
Deschoolmeester
D004247:DNA / Q000302:isolation & purification; D005470:Fluorometry; D020869:Gene Expression Profiling / Q000379:methods*; D006801:Humans; D016612:Paraffin Embedding; D057089:Pathology, Molecular / Q000379:methods*; D012313:RNA / Q000302:isolation & purification*; D011933:Reagent Kits, Diagnostic; D013048:Specimen Handling / Q000379:methods*; D013053:Spectrophotometry; D016707:Tissue Fixation
D011933:Reagent Kits, Diagnostic; D012313:RNA; D004247:DNA