Expression analysis on archival material: comparison of 5 commercially available RNA isolation kits for FFPE material.

Author(s): Boeckx C, Wouters A, Pauwels B, Deschoolmeester V, Specenier P, Lukaszuk K, Vermorken JB, Pauwels P, Peeters M, Lardon F, Baay MF

Publication: Diagn Mol Pathol, 2011, Vol. 20, Page 203-11

PubMed ID: 22089347 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 RNA extraction kit on yield, quality and amplificability from FFPE cervical cancer specimens. FFPE sections had been stored at room temperature for 11.8-13.1 years. RNA was extracted from three 10 uM sections for each of 10-12 specimens. Specimens were deparaffinized with xylene prior to extraction, with the exception of the specimens extracted with QuickExtract which were heated instead.

   Summary of Findings:

   The largest RNA yield and highest purity, as determined by spectrophotometer, was obtained using the RNeasy FFPE Isolation kit followed by the RecoverAll and the ArrayGrade FFPE kits, which had similar yields. The NucleoSpin kit produced low yields, and the authors report that RNA extracted using QuickExtract was contaminated with paraffin. Consequently, RNA extracted with NucleoSpin and QuickExtract were excluded from further analysis. RNA extracted using the RecoverAll kit had a slightly higher average RIN than that extracted with the NucleoSpin, RNeasy or ArrayGrade kits (2.47 versus 2.28, 2.18, and 2.09, respectively). 11 of 12 specimens extracted using RNeasy had sufficient RNA yields for real-time RT-PCR, but only 6 of 12 specimens extracted using RecoverAll and 5 of 12 specimens extracted using ArrayGrade had sufficient yields for real-time RT-PCR analysis. The average CT values for amplification of TBP were similar between RNA extracted with the three different kits, but CT values were over 31 for 20 of the 22 extracts. There were no obvious effects of storage duration on RNA yield.

   Biospecimens

   • Tissue - Cervix

   Preservative Types

   • Formalin

   Diagnoses:

   • Neoplastic - Carcinoma

   Platform:

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

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Storage	Storage duration	11.8-13.1 years
   Real-time qRT-PCR Specific	Targeted nucleic acid	TBP
   Analyte Extraction and Purification	Analyte isolation method	RNeasy FFPE isolation kit RecoverAll Total nucleic acid isolation kit ArrayGrade FFPE RNA isolation kit NucleoSpin FFPE RNA isolation kit QuickExtract FFPE RNA extraction kit

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