RNA extraction from archival formalin-fixed paraffin-embedded tissue: a comparison of manual, semiautomated, and fully automated purification methods.
BACKGROUND
Formalin-fixed paraffin-embedded (FFPE) tumor material represents a valuable resource for the analysis of RNA-based biomarkers, both in research laboratories and in routine clinical testing. A robust and automated RNA-extraction method with a high sample throughput is required.


METHODS
We evaluated extraction performance for 4 silica-based RNA-extraction protocols: (a) a fully automated, bead-based RNA-isolation procedure; (b) its manual counterpart; (c) a semiautomated bead-based extraction system; and (d) a manual column-based extraction kit. RNA from 360 sections (90 sections per extraction method) of 30 FFPE tumor blocks up to 20 years of age was purified and analyzed by quantitative reverse-transcription PCR for ESR1 (estrogen receptor 1), PGR (progesterone receptor), ERBB2 [v-erb-b2 erythroblastic leukemia viral oncogene homolog 2, neuro/glioblastoma derived oncogene homolog (avian)], and RPL37A (ribosomal protein L37a).


RESULTS
The semiautomated protocol gave the best yield. The 3 bead-based methods showed good across-method correlations in both yield and relative mRNA amounts (r = 0.86-0.95 and 0.98, respectively). In contrast, correlations between any of the bead-based methods and the manual column-based method were worse (r = 0.77-0.95 and 0.96, respectively). The fully automated method showed the lowest variation from section to section (root mean square error, 0.32-0.35 Cq, where Cq is the quantification cycle) and required the least hands-on time (1 h).


CONCLUSIONS
The fully automated RNA-purification method showed the best reproducibility in gene expression analyses of neighboring sections of tissue blocks between 3 and 20 years of age and required the least overall and hands-on times. This method appears well suited for high-throughput RNA analyses in both routine clinical testing and translational research studies with archived FFPE material.
Bohmann
Kerstin
K
Siemens Healthcare Diagnostics Products, Molecular Research Germany, Cologne, Germany.
Hennig
Guido
G
Rogel
Uwe
U
Poremba
Christopher
C
Mueller
Berit Maria
BM
Fritz
Peter
P
Stoerkel
Stephan
S
Schaefer
Karl-L
KL
D001331:Automation; D014408:Biomarkers, Tumor / Q000032:analysis / Q000235:genetics; D001943:Breast Neoplasms / Q000737:chemistry / Q000235:genetics; D005557:Formaldehyde; D015870:Gene Expression; D005821:Genetic Techniques; D006801:Humans; D016612:Paraffin Embedding; D012313:RNA / Q000302:isolation & purification*; D015203:Reproducibility of Results; D013997:Time Factors; D016707:Tissue Fixation
D014408:Biomarkers, Tumor; D005557:Formaldehyde; D012313:RNA