Optimization of RNA extraction from FFPE tissues for expression profiling in the DASL assay.

Author(s): Abramovitz M, Ordanic-Kodani M, Wang Y, Li Z, Catzavelos C, Bouzyk M, Sledge GW Jr, Moreno CS, Leyland-Jones B

Publication: Biotechniques, 2008, Vol. 44, Page 417-23

PubMed ID: 18361796 PubMed Review Paper? No

Purpose of Paper

Conclusion of Paper

Studies

1. Study Purpose

   The purpose of this study was to compare RNA quality and yield and DASL assay reproducibility using RNA extracted from FFPE breast cancer specimens with 4 different kits.

   Summary of Findings:

   Increasing proteinase K digestion time to overnight increased RNA yield and DASL assay reproducibility regardless of the kit used. The DASL assay required at least 100 ng RNA, an A260/280 ratio greater than 1.5, and a Rpll13a cycle threshold value less than 29 for an R2 value of greater than 0.9 in 92% of replicates. Only the use of the SuperArray kit with a short proteinase K digestion failed to meet these criteria. The lowest cycle threshold values for Rpl13a and the highest reproducibility were found with the Ambion and Roche kits. The Superarray kit had decreased A260/280 ratio, decreased reproducibility, and higher CT vales. DASL cluster analysis was not altered by RNA preparation method, but did differ between patients.

   Biospecimens

   • Tissue - Breast

   Preservative Types

   • Formalin

   Diagnoses:

   • Neoplastic - Carcinoma

   Platform:

   Analyte	Technology Platform
   RNA	DASL
   RNA	Real-time qRT-PCR
   RNA	Automated electrophoresis/Bioanalyzer

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Analyte Extraction and Purification	Analyte isolation method	HighPure RNA kit (Roche) RecoverAll kit (Ambion) RNeasy FFPE kit (Qiagen) ArrayGrade FFPE RNA isolation kit (SuperArray)
   Analyte Extraction and Purification	Protein digestion	3 h 15 min Overnight

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