Gene expression in formalin-fixed paraffin-embedded lymph nodes.

Author(s): Antica M, Paradzik M, Novak S, Dzebro S, Dominis M

Publication: J Immunol Methods, 2010, Vol. 359, Page 42-6

PubMed ID: 20570676 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 formalin fixation, deparaffinization method, proteinase K digestion duration, and RNA extraction method on the quantity and quality of RNA as determined by spectrophotometer, RT-PCR and real-time PCR. 11 fresh and 18 FFPE lymphoma and tonsil specimens were included. The diagnoses for tonsil specimens and details of the handling of the fresh specimens were not provided. FFPE sections were deparaffinized in three 10 min washes of 56 degrees C xylene or histoclear. Specimens were then placed in TriZol, RNAzol, QIAgen RLT buffer, or in GITC (4 M guanidinium isothiocyanate, 20mM sodium acetate, 0.1 mM dithiothreitol and 0.5% Na-laurylsarcosine, pH 5.5), proteinase K was added, and specimens were digested at 55 degrees C for 1, 3 or 16 h. Formalin cross-links were reversed through incubation at 95 degrees C for 30 min, and proteinase K was inactivated by incubation at 99 degrees C for 5 min. RNA was extracted from the lysates following the protocols of Trizol, RNAZOL, RNeasy, or by phenol chloroform (GITC).

   Summary of Findings:

   Twice as much RNA was obtained from fresh specimens than from FFPE specimens, and RNA from FFPE specimens was of lower quality and generally degraded. The differences in CT values between the Aiolos, Helios and Ikaros transcripts and 18S ribosomal RNA were smaller for FFPE specimens than fresh specimens (p<0.05, p<0.01 and p<0.001, respectively). The authors report that using HistoClear rather than xylene for paraffin removal had no effects on RNA quantity or quality, RNA yield was unaffected by digestion duration, and that the commercial kits provided no benefit over the GITC-phenol chloroform extraction, but data was only shown for the comparison between GITC-phenol chloroform and TriZol.

   Biospecimens

   • Tissue - Lymph Node
   • Tissue - Tonsil

   Preservative Types

   • Formalin
   • None (Fresh)

   Diagnoses:

   • Neoplastic - Lymphoma
   • Not specified

   Platform:

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

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Biospecimen Preservation	Type of fixation/preservation	Formalin (buffered) None (fresh)
   Analyte Extraction and Purification	Protein digestion	1 h 3 h 16 h
   Analyte Extraction and Purification	Analyte isolation method	GITC-phenol chloroform RNAzol RNeasy Trizol
   Analyte Extraction and Purification	Deparaffinization	Xylene HistoClear
   Real-time qRT-PCR Specific	Targeted nucleic acid	Aiolos Helios Ikaros 18S ribosomal RNA

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