Expression analysis on archival material revisited: isolation and quantification of RNA extracted from FFPE samples.

Author(s): Deben C, Zwaenepoel K, Boeckx C, Wouters A, Pauwels P, Peeters M, Lardon F, Baay M, Deschoolmeester V

Publication: Diagn Mol Pathol, 2013, Vol. 22, Page 59-64

PubMed ID: 23370428 PubMed Review Paper? No

Purpose of Paper

Conclusion of Paper

Studies

1. Study Purpose

   The purpose of this study was to determine the effects of using a newer version of the RNeasy FFPE kit which contained DNAse I rather than an older version using gDNA eliminator columns on RNA yield and quality. RNA was extracted from 1-2 10 um FFPE sections of colon, kidney or stomach. The authors do not specify any diagnoses.

   Summary of Findings:

   The yield, ratio of absorbance (A) at 260 to 280 and to 230, and real-time qPCR cycle threshold (CT) values were comparable between RNA isolated from FFPE specimens using two different RNeasy FFPE kits. However, after isolation of RNA with the RNeasy FFPE kit containing the gDNA eliminator columns, 1799.5 ng DNA remained in the specimen, but after isolation of RNA with the RNeasy FFPE kit containing DNAse I, only 958.4 ng DNA remained.

   Biospecimens

   • Tissue - Colorectal
   • Tissue - Stomach
   • Tissue - Kidney

   Preservative Types

   • Formalin

   Diagnoses:

   • Not specified

   Platform:

   Analyte	Technology Platform
   RNA	Fluorometry
   RNA	Spectrophotometry
   RNA	Real-time qRT-PCR

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Analyte Extraction and Purification	Analyte isolation method	RNeasy FFPE kit with gDNA eliminator column RNeasy FFPE kit with DNAse I
   Analyte Extraction and Purification	Nucleic acid digestion	DNAse I Eliminator column
   Real-time qRT-PCR Specific	Targeted nucleic acid	Hydroxymethylbilane synthase

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2. Study Purpose

   The purpose of this study was to compare measurement of RNA from FFPE colorectal tumors with the Nanodrop spectrophotometer and with the Qubit 2.0 Fluorometer.

   Summary of Findings:

   The concentration of RNA extracted from FFPE specimens was, on average, 32.03% higher when measured by the Nanodrop spectrophotometer than when measured by the Qubit 2.0 fluorometer, but this difference could be eliminated by 1:4 or 1:8 dilution of RNA in water before spectrophotometric measurements.

   Biospecimens

   • Tissue - Colorectal

   Preservative Types

   • Formalin

   Diagnoses:

   • Neoplastic - Carcinoma

   Platform:

   Analyte	Technology Platform
   RNA	Fluorometry
   RNA	Spectrophotometry

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Biospecimen Aliquots and Components	Biospecimen components	Undiluted Diluted 1:2 Diluted 1:4 Diluted 1:8
   Spectrophotometry Specific	Technology platform	Qubit 2 fluorometer

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