RNA extraction from archival formalin-fixed paraffin-embedded tissue: a comparison of manual, semiautomated, and fully automated purification methods.
Author(s): Bohmann K, Hennig G, Rogel U, Poremba C, Mueller BM, Fritz P, Stoerkel S, Schaefer KL
Publication: Clin Chem, 2009, Vol. 55, Page 1719-27
PubMed ID: 19617290 PubMed Review Paper? No
Purpose of Paper
Conclusion of Paper
Studies
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Study Purpose
The purpose of this study was to determine the effects of specimen age and extraction method on RNA yield and quantification from FFPE breast tumor specimens.
Summary of Findings:
Real-time PCR amplification was possible in all specimens which had been fully extracted. Using CT values as an indicator of yield, method 3 (CT=22.7) performed the best followed by the RNeasy FFPE kit (method 4)( CT=22.8), the manual VERSANT kPCR with extra centrifugation (method 2)(CT=22.9) and method 1 (CT=23.3). All methods produced two clusters of CT values. The higher CT group (lower yield) corresponded to specimens that were 20 years old. Within this group, method 4 had a substantially lower RNA yield. When breast cancer marker genes were examined, the normalized values between methods were much better correlated than the CTs. While method 3 had the best yield, method 1 gave the most reproducible results making it the method of choice.
Biospecimens
Preservative Types
- Formalin
Diagnoses:
- Neoplastic - Carcinoma
Platform:
Analyte Technology Platform RNA Real-time qRT-PCR Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Storage Storage duration 3 years
5 years
20 years
Analyte Extraction and Purification Analyte isolation method Automated VERSANT kPCR molecular system (Siemens Healthcare)
Manual VERSANT kPCR with extra centrifugation molecular system (Siemens Healthcare)
EZI RNA tissue Minikit (Qiagen)
RNeasy FFPE kit (Qiagen)