A simple method for RNA isolation from formalin-fixed and paraffin-embedded lymphatic tissues.

Author(s): Körbler T, Grskovic M, Dominis M, Antica M

Publication: Exp Mol Pathol, 2003, Vol. 74, Page 336-40

PubMed ID: 12782023 PubMed Review Paper? No

Purpose of Paper

Conclusion of Paper

Studies

1. Study Purpose

   The purpose of this study was to optimize RNA isolation from formalin-fixed, paraffin-embedded (FFPE) lymphatic specimens for subsequent RT-PCR analysis of three genes: beta-actin, Ikaros, Aiolos.

   Summary of Findings:

   The authors noted optimal RT-PCR results for the three genes investigated with amplicons ranging in size from 194 to 374 bp when FFPE specimens were deparaffinized in xylene for 5 minutes at 57 degrees C, extracted in buffers lacking RNAse inhibitors (particularly the pH 7.6 buffer comprising NaCl, Tris, EDTA, and SDS), and incubated with proteinase K at 45 degrees overnight followed by necessary inactivation for 7 minutes at 100 degrees C. Inclusion or omission of a rehydration step following deparaffinization did not impact RT-PCR results.

   Biospecimens

   • Tissue - Lymph Node

   Preservative Types

   • Formalin

   Diagnoses:

   • Neoplastic - Lymphoma

   Platform:

   Analyte	Technology Platform
   RNA	RT-PCR

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Analyte Extraction and Purification	Deparaffinization	Xylene Histoclear Post-deparaffinization rehydration step
   Analyte Extraction and Purification	Analyte isolation method	Guanidium isothiocyanate buffer, pH 5.5 NaCl/Tris/EDTA/SDS buffer, pH 7.6 NaCl/Tris/EDTA/SDS buffer, pH 8.0 Tris/EDTA/SDS buffer, pH 8.0
   Analyte Extraction and Purification	Protein digestion	Proteinase K (3 h, 55 degrees C) Proteinase K (overnight, 45 degrees C) Proteinase K inactivation (7 min, 100 degrees C)

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2. Study Purpose

   The purpose of this study was to compare the quality of RNA extracted from FFPE lymphatic specimens with freshly isolated specimens.

   Summary of Findings:

   Quality of extracted RNA, determined by electrophoresis of total RNA, was compromised among FFPE specimens compared to fresh specimens, with smears versus distinct ribosomal bands observed, respectively. While a quantitative comparison of RT-PCR results was not performed among differentially fixed specimens the authors conclude that RT-PCR based screening of FFPE specimens was successful using the modified extraction technique.

   Biospecimens

   • Tissue - Lymph Node

   Preservative Types

   • Formalin
   • None (Fresh)

   Diagnoses:

   • Neoplastic - Lymphoma

   Platform:

   Analyte	Technology Platform
   RNA	RT-PCR
   RNA	Electrophoresis

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Biospecimen Preservation	Type of fixation/preservation	Formalin (buffered) None (fresh)

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